Genetically encoded biosensors

ABSTRACT

The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/904,574filed Feb. 26, 2018, which is a divisional of U.S. application Ser. No.15/664,326 filed Jul. 31, 2017, which is a divisional of U.S.application Ser. No. 14/350,199, filed Nov. 18, 2014, which is a 35U.S.C. § 371 of International Application No. PCT/US2012/059219, filedOct. 8, 2012, which claims priority to U.S. Application No. 61/544,867filed Oct. 7, 2011.

TECHNICAL FIELD

This disclosure relates to genetically encoded biosensors and methodsfor the design, production, and use of such biosensors.

BACKGROUND

Protein-based sensors that transduce microscopic binding events intomacroscopically observable signals are available to allow real-timevisualization of a variety of biological events and/or molecules(Frommer et al., Chem. Soc. Rev., 38:2833-2841, 2009). Such sensors canbe targeted and/or expressed in living cells, tissues, and organisms,and permit imaging with minimally invasive techniques (Okumoto, Curr.Opin. Biotechnol., 21:45-54, 2010). Application of these sensors islimited by the narrow range of analytes that can be detected and/or bytheir inability to distinguish signal over noise.

SUMMARY

The present disclosure provides genetically encoded recombinant peptidescontaining an analyte-binding framework portion linked (e.g., operablylinked) to a signaling portion, wherein the signaling portion isallosterically regulated by the framework portion upon interaction ofthe framework portion with an analyte (e.g., a defined, selected, and/orspecific analyte). These constructs can be used as biosensors, e.g., totransduce microscopic binding events into macroscopically observablesignals.

The present disclosure provides, in part, recombinant peptides for useas biosensors (e.g., recombinant peptide biosensors) that include (e.g.,comprise, consist essentially of, or consist of), e.g., include atleast, an analyte-binding framework portion and a signaling portion. Asdescribed in further detail herein, such signaling portions are presentwithin the framework portion at a site or amino acid position thatundergoes a conformational change (e.g., a conformational changesufficient to alter a physical and/or functional characteristic of thesignaling portion, e.g., a substantial conformational change) uponinteraction of the framework portion with a defined, specific, orselected analyte (e.g. such as an analyte to which the framework portionor a region thereof, and/or the biosensor, specifically binds). Forexample, in some instances, the signaling portion is allostericallyregulated by the framework portion such that signaling from thesignaling portion is altered (e.g. wherein a first level of signaling isaltered or changed to a second level of signaling that can bedistinguished using routine methods of detection from the first) uponinteraction of the framework portion with the analyte. In someinstances, signaling by the signaling portion can detectably increase ordecrease upon interaction of the framework portion with the analyte. Insome instances, signaling by the signaling portion upon interaction ofthe biosensor with a defined, specific, or selected analyte (e.g. suchas an analyte to which the framework portion or a region thereof, and/orthe biosensor, specifically binds) can be proportional or can correlatewith to the level of interaction between the framework portion and theanalyte such that the level of interaction can be determined from thesignaling or alteration thereof.

In some instances, framework portions of the biosensors disclosed hereinhave a first structure in the absence of an analyte and a secondstructure that is detectably distinct from the first structure in thepresence of the analyte. In some instances, the conformational changebetween the first structure and the second structure allostericallyregulates the signaling portion.

In some instances, framework portions of the biosensors disclosed hereincan be, or can include (e.g., comprise, consist essentially of, orconsist of), periplasmic binding proteins (PBP) or variants of a PBP. Insome instances, exemplary PBPs or variants thereof can include, but arenot limited to, peptides with at least 90% identity to a peptideselected from the group consisting of SEQ ID NO:105, SEQ ID NO: 106, SEQID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO:111,SEQ ID NO:113, and SEQ ID NO:114. In some instances, exemplary PBPs orvariants thereof can include, but are not limited to, peptides with atleast 95% identity to a peptide selected from the group consisting ofSEQ ID NO:105, SEQ ID NO: 106, SEQ ID NO:107, SEQ ID NO:108, SEQ IDNO:109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO:113, and SEQ ID NO:114.In some instances, exemplary PBPs or variants thereof can include, butare not limited to, peptides selected from the group consisting of SEQID NO:105, SEQ ID NO: 106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109,SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO:113, and SEQ ID NO:114. In someinstances, exemplary PBPs or variants thereof can include, but are notlimited to, peptides selected from the group consisting of SEQ IDNO:105, SEQ ID NO: 106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQID NO: 110, SEQ ID NO:111, SEQ ID NO:113, and SEQ ID NO:114 comprising10 or fewer conservative amino acid substitutions. PBPs or variantsthereof disclosed herein can be truncated.

In some instances, signaling portions of the biosensors disclosed hereincan be or can include (e.g., comprise, consist essentially of, orconsist of) one or more (e.g., one, two three, four, five, and less thanten) circularly permuted fluorescent proteins (cpFPs). Such cpFPs can beinclude but are not limited to, for example, green fluorescent proteins,yellow fluorescent proteins, red fluorescent proteins, and/or bluefluorescent proteins.

In some instances, biosensors disclosed herein, e.g., analyte-bindingframework portions of biosensors disclosed herein, can bind (e.g., bindspecifically) to glucose. Such sensors can be referred to as glucosebinding biosensors or glucose biosensors.

In some instances, biosensors disclosed herein, e.g., analyte-bindingframework portions of biosensors disclosed herein, can bind (e.g., bindspecifically) to maltose. Such sensors can be referred to as maltosebinding biosensors or maltose biosensors.

In some instances, biosensors disclosed herein, e.g., analyte-bindingframework portions of biosensors disclosed herein, can bind (e.g., bindspecifically) to phosphonate. Such sensors can be referred to asphosphonate binding biosensors or phosphonate biosensors.

In some instances, biosensors disclosed herein, e.g., analyte-bindingframework portions of biosensors disclosed herein, can bind (e.g., bindspecifically) to glutamate. Such sensors can be referred to as glutamatebinding biosensors or glutamate biosensors.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): an amino acid sequencewith at least 90% identity to a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51,52, and 53, wherein the recombinant peptide biosensor binds specificallyto maltose; a recombinant peptide biosensor selected from the groupconsisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and 53comprising 10 or fewer conservative amino acid substitutions, whereinthe recombinant peptide biosensor binds specifically to maltose; and/ora recombinant peptide biosensor selected from the group consisting ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and 53.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): an amino acid sequencewith at least 90% identity to a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 62 and 63, wherein therecombinant peptide biosensor binds specifically to glutamate; arecombinant peptide biosensor selected from the group consisting of SEQID NO: 62 and 63 comprising 10 or fewer conservative amino acidsubstitutions, wherein the recombinant peptide biosensor bindsspecifically to glutamate; and/or a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 62 and 63.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): an amino acid sequencewith at least 90% identity to a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 77 and 78, wherein therecombinant peptide biosensor binds specifically to phosphonate; arecombinant peptide biosensor selected from the group consisting of SEQID NO: 77 and 78 comprising 10 or fewer conservative amino acidsubstitutions, wherein the recombinant peptide biosensor bindsspecifically to phosphonate; and/or a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 77 and 78.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): an amino acid sequencewith at least 90% identity to a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 91, 92, 93 and 94, wherein therecombinant peptide biosensor binds specifically to glucose; arecombinant peptide biosensor selected from the group consisting of SEQID NO: 91, 92, 93 and 94 comprising 10 or fewer conservative amino acidsubstitutions, wherein the recombinant peptide biosensor bindsspecifically to glucose; and/or a recombinant peptide biosensor selectedfrom the group consisting of SEQ ID NO: 91, 92, 93 and 94.

In some instances, biosensors disclosed herein can include (e.g.,comprise, consist essentially of, or consist of): SEQ ID NO:91; SEQ IDNO:92; SEQ ID NO:93; SEQ ID NO:95.

In some instances, any recombinant biosensor disclosed herein can beisolated and/or purified. The terms “isolated” or “purified,” whenapplied to a biosensor disclosed herein includes nucleic acid proteinsand peptides that are substantially free or free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free or free of precursors or other chemicals whenchemically synthesized.

The disclosure also provides, in part, nucleic acids (e.g., isolatedand/or purified nucleic acids) encoding any one or more of therecombinant peptide biosensors disclosed herein. For example, nucleicacids can encode: an amino acid sequence with at least 90% identity to arecombinant peptide biosensor selected from the group consisting of SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and 53, wherein therecombinant peptide biosensor binds specifically to maltose; arecombinant peptide biosensor selected from the group consisting of SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and 53 comprising 10 or fewerconservative amino acid substitutions, wherein the recombinant peptidebiosensor binds specifically to maltose; a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,50, 51, 52, and 53; an amino acid sequence with at least 90% identity toa recombinant peptide biosensor selected from the group consisting ofSEQ ID NO: 62 and 63, wherein the recombinant peptide biosensor bindsspecifically to glutamate; a recombinant peptide biosensor selected fromthe group consisting of SEQ ID NO: 62 and 63 comprising 10 or fewerconservative amino acid substitutions, wherein the recombinant peptidebiosensor binds specifically to glutamate; a recombinant peptidebiosensor selected from the group consisting of SEQ ID NO: 62 and 63; anamino acid sequence with at least 90% identity to a recombinant peptidebiosensor selected from the group consisting of SEQ ID NO: 77 and 78,wherein the recombinant peptide biosensor binds specifically tophosphonate; a recombinant peptide biosensor selected from the groupconsisting of SEQ ID NO: 77 and 78 comprising 10 or fewer conservativeamino acid substitutions, wherein the recombinant peptide biosensorbinds specifically to phosphonate; a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 77 and 78; an aminoacid sequence with at least 90% identity to a recombinant peptidebiosensor selected from the group consisting of SEQ ID NO: 91, 92, 93and 94, wherein the recombinant peptide biosensor binds specifically toglucose; a recombinant peptide biosensor selected from the groupconsisting of SEQ ID NO: 91, 92, 93 and 94 comprising 10 or fewerconservative amino acid substitutions, wherein the recombinant peptidebiosensor binds specifically to glucose; a recombinant peptide biosensorselected from the group consisting of SEQ ID NO: 91, 92, 93 and 94;and/or SEQ ID NO:91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:95.

In some instances, the disclosure includes vectors containing one or aplurality of the nucleic acids disclosed herein and cells containingsuch vectors. In some instances, the disclosure provides cellscontaining one or a plurality of nucleic acids disclosed herein.

In some instances, the disclosure includes kits related to thebiosensors and nucleic acids disclosed herein. Such kits can include orcontain, for example, a biosensor, a nucleic acid encoding a biosensor,vectors, and/or cells, provided herein.

In some instances, the disclosure provides methods related to thebiosensors and nucleic acids disclosed herein. Such methods can includemethods of making, using, and/or selling the biosensors and nucleicacids disclosed herein. For example, methods can include methods forproducing genetically encoded recombinant peptide biosensors. In suchinstances, methods can include, for example, selecting a frameworkportion that binds specifically to a target analyte and that undergoes aconformational change upon interacting binding to the target analyte,identifying a site or amino acid position within the selected frameworkportion where or around which the conformational change occurs, andinserting a signaling portion into the site or amino acid position. Insome instances, framework portions include periplasmic binding proteins(PBPs) disclosed herein. Exemplary PBPs include PBPs that bind (e.g.,bind specifically) to glucose.

In some instances, the present disclosure includes methods for detectingglucose, e.g., in a sample containing a level of glucose. Such methodscan include, detecting a level of fluorescence emitted by a recombinantpeptide biosensor, the peptide biosensor having an amino acid sequenceselected from the group consisting of SEQ ID NO: 91, 92, 93 and 94, andcorrelating the level of fluorescence with the presence of glucose. Insome instances, recombinant peptide biosensors used in the methodsherein are expressed from nucleic acids. In some instances, methodsinclude contacting the recombinant peptide biosensor with a test sample(e.g., a sample comprising glucose). In some instances, methods caninclude the level of fluorescence emitted by a biosensor (e.g., abiosensor bound to glucose) with a concentration glucose in the sample.Such correlation can include, for example, comparing the level offluorescence with a level of fluorescence emitted by the recombinantpeptide biosensor in the presence of a sample comprising a knownconcentration or range of concentrations of glucose. In some instance,the level of fluorescence emitted by the recombinant peptide biosensorin the presence (e.g., bound or bound specifically to) of a samplecomprising a known concentration or range of concentrations of glucoseis stored on an electronic database.

One of skill will appreciate that such methods can be adapted for anydefined, specific, or selected analyte. For example, in some instances,the disclosure provides methods for detecting a defined, selected, orspecific analyte. These methods can include detecting a level offluorescence emitted by a recombinant peptide biosensor expressed from anucleic acid and correlating the level of fluorescence with the presencethe defined, selected, or specific analyte. In some instances, methodsinclude contacting the recombinant peptide biosensor with a samplecomprising the analyte. In some instances, methods include correlatingthe level of fluorescence with a concentration of the analyte. In someinstances, methods include comparing the level of fluorescence with alevel of fluorescence emitted by the recombinant peptide biosensor inthe presence of a sample comprising a known concentration or range ofconcentrations of the analyte, wherein the level of fluorescence emittedby the recombinant peptide biosensor in the presence of a samplecomprising a known concentration or range of concentrations of theanalyte is stored on an electronic database.

In some instances, the present disclosure provides methods for detectinga defined, selected, or specific analyte, the method comprisingdetecting a level of fluorescence emitted by a recombinant peptidebiosensor as described herein; and correlating the level of fluorescencewith the presence of a defined, selected, or specific analyte. In someinstances, recombinant peptide biosensors can be expressed from anucleic acid. In some instances, methods can include contacting therecombinant peptide biosensor with a sample comprising the analyte. Insome instances, methods can include correlating the level offluorescence with a concentration of the analyte and, optionally,comparing the level of fluorescence with a level of fluorescence emittedby the recombinant peptide biosensor in the presence of a samplecomprising a known concentration or range of concentrations of theanalyte. In some instances, the level of fluorescence emitted by therecombinant peptide biosensor in the presence of a sample comprising aknown concentration or range of concentrations of the analyte is storedon an electronic database.

Methods herein can be performed in vitro.

In some instances, the present disclosure provides compositionscontaining any one or a plurality of the peptide biosensors and/ornucleic acids disclosed herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1|Cartoon representation showing ligand bound Escherichia Colimalto-dextrin-binding protein (EcMBP) and potential circularly-permutedfluorescent protein (cpFP) insertion sites.

FIG. 2|Cartoon representation showing ligand bound Pyrococcus furiosusmaltotriose binding protein (PfMBP) and potential cpFP insertion sites.

FIG. 3|Cartoon representation showing ligand bound E. coliglutamate-binding protein (EcYbeJ) and potential cpFP insertion sites.

FIG. 4|Cartoon representation showing ligand bound E. coliphosphonate-binding protein (EcPhnD) and potential cpFP insertion sites.

FIG. 5|Cartoon representation showing ligand bound Thermus thermophilusglucose binding protein (TtGBP) and potential cpFP insertion sites.

FIG. 6A-B|Changes in EcMBP upon maltose binding and locations at whichcircularly-permuted fluorescent protein (cpFP) was inserted are shown ascolored spheres at the Cα positions. Yellow: 165-166, Green: 175-176,Cyan: 311-312, Violet: 317-318(A). (B) shows backbone structuralchanges. The Cα dihedral is calculated from the four atoms: Cαi+2,Cαi+1, Cαi, Cαi−1. ΔDihedral is calculated as the difference indihedrals between the closed (1ANF) and open (1OMP) states of MBP, andcorrected to fall within a range of −180° to 180°. The regions nearresidues 175 and 311 are labeled. There is a crystallographic artifactat the N-terminus resulting in the appearance of significant structuralchanges.

FIG. 7A|Amino acid sequence of MBP-165-cpGFP (SEQ ID NO:1).

FIG. 7B|Amino acid sequence of MBP-165-cpGFP.PPYF (SEQ ID NO:2).

FIG. 7C|Amino acid sequence of MBP-165-cpGFP.PCF (SEQ ID NO:3).

FIG. 8A|Amino acid sequence of MBP-175-cpGFP (SEQ ID NO:4).

FIG. 8B|Amino acid sequence of MBP-175-cpGFP.L1-HL (SEQ ID NO:5).

FIG. 9A|Amino acid sequence of MBP-311-cpGFP (SEQ ID NO:6).

FIG. 9B|Amino acid sequence of MBP-311-cpGFP.L2-NP (SEQ ID NO:7).

FIG. 10|Amino acid sequence of MBP-317-cpGFP (SEQ ID NO:8).

FIGS. 11A-11D|Line charts showing EcMBP plot of ΔF/F for clarifiedlysate screen of cpGFP linker-screens at insertion points 165, 175, 311,and 317. The horizontal dashed line at zero indicates no fluorescencechange. Standard deviations in ΔF/F are less than 10% of an average ΔF(repetitions for MBP165-cpGFP.PPYF yields ΔF/F values of 2.51, 2.63, and2.54).

FIG. 12|Isothermal titration calorimetry (ITC) of MBP317-cpGFP withmaltose.

FIG. 13|Graph showing EcMBP165-cpGFP.PPYF affinity variant bindingmaltose-binding curves. Binding curves for affinity variants ofMBP165-cpGFP.PPYF. Data is fit to a single-binding site isotherm.Curve-fit affinities are: WT binding pocket, 5 μM (●); W230A, 32 μM (▪);W62A, 375 μM (▴); W340A, >1 mM (▾); 1329W, 11 μM (□).

FIGS. 14A-14D|Line graphs showing maltose and sucrose binding curves forwild-type and 5-7 variants of the EcMBP-cpGFP sensors. Maltose (black)and sucrose (red) binding curves for wild-type (filled, solid lines) and5-7 variants (open, dashed lines) of the MBP-cpGFP sensors.MBP165-cpGFP.PPYF (a); MBP165-cpGFP.PCF (b); MBP175-cpGFP.L1-HL (c);MBP311-cpGFP.L2-NP (d).

FIGS. 15A-15D|Line graphs showing emission spectra for colored variantsof EcMBP sensors. Fluorescence emission spectra of the MBP165-Blue,Cyan, Green, and Yellow wild-type sensors (a) and the 5-7 variants (b)in the absence of ligand (dashed lines, open circles), with 10 mMmaltose (solid lines, filled circles), or 10 mM sucrose (solid lines,filed squares). Sensors were excited at 383, 433, 485, and 485 nm,respectively. Titration of maltose and sucrose in the Blue, Cyan, Green,and Yellow MBP165 wild-type sensors (c) and for the 5-7 variants (d).Filled circles are titration of maltose, open circles are titration ofsucrose. For the wild-type sensors, Kds for maltose binding are: Blue3.3 μM, Cyan 13 μM, Green 4.5 μM, Yellow 3.3 μM. No sucrose binding isobserved. For the 5-7 variants, Kd of Green is 2.4 mM (sucrose) and 7.1mM (maltose). Kd of Yellow is 2.5 mM (sucrose) and 4.5 mM (maltose).

FIG. 16|Plot of ΔF/F for clarified lysate screen of MBP165-cpBFPlinker-screen. The horizontal dashed line at zero indicates nofluorescence change.

FIGS. 17A-17B|Line graphs showing maltose binding. Blue (wt bindingpocket) has an affinity of 2.7 μM. Green (W230A) has an affinity of 40μM. Yellow (W62A) has an affinity of 350 μM. Cyan (W340A) has anaffinity of approximately 1.7 mM. Data is plotted at ΔF/F (a) ornormalized to Fractional Saturation (b).

FIGS. 18A-18C|Images bacterial cells expressing (a) EGFP, (b) PPYF, or(c) PPYF.T203V in the absence (top) and presence (bottom) of maltose.

FIGS. 19A-19B|Line graphs showing EcMBP-cpGFP.PPYF.T203V 2-photonexcitation spectra. MBP165-cpAzurite.L2-FE (a), -cpCFP.PCF (a),-cpGFP.PPYF (b), and -cpYFP.PPYF (b) were excited at the wavelengthsindicated and emission measured through appropriate wavelength filters.Two graphs are shown to present different y-axis scales. Optimal ΔF/Fvalues for 2-photon excitation of the spectral variants of MBP165 are:-cpAzurite, 1.1 (ex 760 nm); -cpCFP, 2.3 (ex 830-960 nm); -cpGFP, 10.0(ex 940 nm); -cpYFP, 2.6 (ex 940 nm).

FIGS. 20A-20C|Images showing EcMBP-cpGFP.PPYF.T203V expressing HEKcells. Images of individual HEK293 cells expressing membrane displayedPPYF.T203V in the absence of maltose (a), in the presence of 1 mMmaltose (b), and after washout with maltose-free buffer (c). Scale barsare 10 μm.

FIGS. 21A-21B|Graphs showing quantification of fluorescence ofEcMBP-cpGFP.PPYF.T203V when displayed on the surface of HEK cells. (a)Concentration dependence. (b) Observed fluorescence after a “puff” ofHBSS solution containing 1 mM maltose and 2.5 nM Alexa Fluor® 568(Invitrogen, Carlsbad, Calif.).

FIGS. 22A-22D|Cartoon representations and close-up views of inter-domainlinkers and selected amino acids of the cpGFP chromophore environment ofthe structure of MBP175-cpGFP.L1-HL (A and B) and MBP311-cpGFP.L2-NP (Cand D) bound to maltose. The MBP domain is colored as in FIG. 1. ThecpGFP domain is green and the inter-domain linkers are colored white.The cpGFP chromophore is displayed as sticks and the bound maltose asred and white spheres. Ordered water molecules are represented as redspheres. Selected hydrogen bonds are displayed as dashed black lines.β-strands 10 and 11 of cpGFP are displayed as semi-transparent forclarity. The 2Fo-Fc electron density map calculated with the displayedresidues omitted from the model is shown as blue mesh.

FIGS. 23A-23D|EcMBP-cpGFP: effect of T203V mutation on fluorescence. (a)Emission spectra of 1 μM purified eGFP (filled circles), cpGFP (filledsquares), MBP165-cpGFP.PPYF (open circles), and MBP165-cpGFP.PPYF+T203V(open squares) in the absence (dashed lines) or presence (solid lines)of 1 mM maltose. cpGFP is half as bright as eGFP, and the saturatedMBP165-cpGFP.PPYF variants are about half as bright as cpGFP. (b)Titration of maltose for MBP165-cpGFP.PPYF (filled squares), andMBP165-cpGFP.PPYF+T203V (filled circles). Affinities for each proteinare the same, but with different ΔF/F. (c) Emission spectra of 1 μMpurified eGFP (filled circles), cpGFP (filled squares),MBP311-cpGFP.L2-NP (open circles), and MBP311-cpGFP.L2-NP+T203V (opensquares) in the absence (dashed lines) or presence (solid lines) of 1 mMmaltose. Note that mutation T203V decreases the fluorescence of both theapo-state and the saturated state of MBP311-cpGFP.L2-NP. (d) Titrationof maltose for MBP311-cpGFP.L2-NP (filled squares), andMBP311-cpGFP.L2-NP+T203V (filled circles). Affinities for each proteinare the same, but with ΔF/F slightly increased for the T203V variant.

FIG. 24A|Amino acid sequence of PfMBP171-cpGFP (SEQ ID NO:50)

FIG. 24B|Amino acid sequence of PfMBP171cpGFP.L2-FE (SEQ ID NO:51)

FIG. 25A|Amino acid sequence of PfMBP316-cpGFP (SEQ ID NO:52)

FIG. 25B|Amino acid sequence of PfMBP316-cpGFP.L1-NP (SEQ ID NO:53)

FIG. 26A-26B|Plot of ΔF/F for clarified lysate screen of cpGFPlinker-screens at insertion points 171 (A) and 316 (B).

FIGS. 27A-27D|Plot of Beta-sheet circular dichroism (CD) signal as afunction of temperature.

FIGS. 28A-28B|PfMBP Fluorescence vs. temperature. (A) Plot offluorescence as a function of temperature in the presence (solid) orabsence (dashed) of ligand. (B) Plot of ΔF/F as a function oftemperature. Using the data from panel (a), ΔF/F for each protein(Fbound-Fapo/Fapo) was calculated for each temperature.

FIGS. 28C-28E|Line graphs showing the function of immobilized andsoluble proteins.

FIG. 29A|Amino acid sequence of EcYbeJ253-cpGFP (SEQ ID NO:62).

FIG. 29B|Amino acid sequence of EcYbeJ253-cpGFP.L1LVL2NP (SEQ ID NO:63).

FIG. 30|EcYbeJ binding curves. Plot of ΔF/F as a function of[Glutamate], μM. The first generation sensor, EcYbeJ253.L1-LV (with theA184V) mutation (grey, solid) has an affinity for glutamate of about 100μM and a ΔF/F of 1.2. The reversion of that affinity mutation, V184A, inthe L1-LV background increases affinity to 1 (grey dashed). The secondgeneration sensor, with the L2-NP linker optimization and the A184Vmutation, has a ΔF/F of at least 4 and an affinity for glutamate ofabout 100 μM (black solid).

FIG. 31|EcYbeJ Hema/cMyc analysis. The effect of N- and C-terminal tagson ΔF/F and glutamate affinity were determined by expressing variouslytagged versions of the EcYbeJ253.L1LVL2NP protein in bacteria. Thepresence of the pRSET leader sequence (black) has no effect on ΔF/F (˜5)or affinity (˜120 when compared to the version without a tag (grey). Theaddition of the cMyc tag to the C-terminus retains ΔF/F and increasesaffinity slightly, to 60 μM. The addition of the N-terminalhemagglutinin tag, with (green) or without (orange) the cMyc tag,decreases ΔF/F substantially.

FIGS. 32A-32B|EcYbeJ253-cpGFP.L1LVL2NP.pMinDis expressed in HEK293cells. (A) Images of the sensor expressing HEK cells in the absence ofglutamate (left), with 100 μM glutamate (center), and re-imaged afterwash-out of glutamate with buffer (right). (B) By measuring theequilibrium ΔF/F with different concentrations of glutamate in thebuffer, an in situ binding affinity (black) can be obtained. The surfacedisplayed sensor has a higher affinity (3 μM) for glutamate than thesoluble sensor (grey), which is about 90 μM.

FIG. 33|EcYbeJ253-cpGFP.L1LVL2NP.pMinDis expressed in neuronal culture,and responds rapidly to added glutamate (green). Red shows signal of 2.5nM Alexa Fluor® 568 (Invitrogen, Carlsbad, Calif.), also in pipette.

FIG. 34A|Amino acid sequence of EcPhnD90-cpGFP (SEQ ID NO:77).

FIG. 34B|Amino acid sequence of EcPhnD90-cpGFP.L1AD+L297R+L301R (SEQ IDNO: 78).

FIGS. 35A-35C|EcPhnD90-cpGFP Binding Curves. For both the L1AD and theL1AD+L297R+L301R variants, binding was determined for (A)2-aminoethylphosphonate (2AEP), (B) methylphosphonate (MP), and (C)ethylphosphonate (EP).

FIGS. 36A-36C|The crystal structures of the ligand-free (A), open state(with H157A mutation to the binding pocket) and the ligand-bound (B),closed state of EcPhnD clearly shows a large conformational change.Residues in between which cpGFP is inserted in EcPhnD90-cpGFP are markedby red spheres, in the equatorial strand (red). (C) Analysis of thechange in Cα dihedral (ΔDihedral) clearly shows that residues for whichthere is the greatest ΔDihedral upon going from the open to the closedstate are residues 88 (ΔDihedral=−75°), 89 (ΔDihedral=123°), and 90(ΔDihedral=52°).

FIG. 37A|Amino acid sequence of TtGBP326-cpGFP (SEQ ID NO:91).

FIG. 37B|Amino acid sequence of TtGBP326.L1-PA (SEQ ID NO:92).

FIG. 37C|Amino acid sequence of TtGBP326.H66A (SEQ ID NO:93).

FIG. 37D|Amino acid sequence of TtGBP326.H348A (SEQ ID NO:94).

FIG. 38|TtGBP326-cpGFP Binding Curves. Plot of ΔF/F as a function of[Glucose], mM.

FIG. 39|An image showing TtGBP326-cpGFP expressed as a transgenicreporter of intracellular glucose in cultured human cells.

FIGS. 40A-40B|Are line graphs showing that the addition of extracellularglucose increases TtGBP326-cpGFP fluorescence in human cells.

FIG. 41|Amino acid sequence of Escherichia coli maltodextrin-bindingprotein (EcMBP) (SEQ ID NO: 105).

FIG. 42|Amino acid sequence of Pyrococcus furiosus maltose-bindingprotein (PfMBP) (SEQ ID NO: 106).

FIG. 43|Amino acid sequence of E. coli glutamate-binding protein(EcYbeJ) (SEQ ID NO:107).

FIG. 44|Amino acid sequence of E. coli phosphonate-binding protein(EcPhnD) (SEQ ID NO:108).

FIG. 45|Amino acid sequence of Thermus thermophilus glucose-bindingprotein (TtGBP) (SEQ ID NO:109).

FIG. 46|Amino acid sequence of UniProt accession number Q92N37 (SEQ IDNO: 110).

FIG. 47|Amino acid sequence of UniProt accession number D0VWX8 (SEQ IDNO:111).

FIG. 48|Amino acid sequence of UniProt accession number Q7CX36 (SEQ IDNO:112).

FIG. 49|Amino acid sequence of UniProt accession number P0AD96 (SEQ IDNO:113).

FIG. 50|Amino acid sequence of TtGBP326.L1PA.L2NP.H66A.H348A.L276V (SEQID NO:114).

FIG. 51|A line graph showing binding ofTtGBP326.L1PA.L2NP.H66A.H348A.L276V to glucose.

FIG. 52|A line graph showing fluorescence increase upon addition ofglucose to HEK293 cells expressing TtGBP326.LIPA.L2NP.H66A.H348A.L276Von their extracellular surface.

FIG. 53|A schematic of Structure I as described herein.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the discovery ofstructures and methods related to and useful for genetically encodedbiosensors. Specifically, the disclosure provides genetically encodedrecombinant or chimeric peptides for use as biosensors and methods forthe design, production, and use of such biosensors. As described below,these sensors can be employed (e.g., expressed) in biological systems todetect and/or monitor a wide range of target analytes (e.g., a defined,selected, and/or specific analytes) due, in part, to the signal changegenerated by the sensors upon binding to their respective analyte(s),which signal change allows bound and unbound sensors to bedistinguished.

While the disclosure encompasses generic biosensors and methods relatedthereto, examples of particular binding sensors, including biosensorsfor detecting maltose, sucrose, maltotriose, glutamate, phosphonate, andglucose are also disclosed.

Compositions

Provided herein are genetically encoded biosensors, i.e., nucleic acidsencoding peptides, and/or the encoded peptides (e.g., isolatedpeptides), for use as biosensors. Biosensors herein include geneticallyencoded recombinant peptides containing an analyte-binding frameworkportion linked (e.g., operably linked) to at least one independentsignaling portion, wherein the independent signaling portion isallosterically modulated or regulated by the framework portion uponinteraction of the framework portion with an analyte (e.g., a defined,selected, and/or specific analyte), such that signaling from thesignaling portions is altered upon interaction of the framework portionwith the analyte.

In some instances, an independent signaling portion is present at a sitewithin the framework portion that undergoes a conformational change uponinteraction of the framework portion with an analyte such that theconformational change allosterically modulates or regulates signaling bythe signaling portion. For example, biosensors herein can includestructure I (FIG. 53).

As described herein, the signaling portion is present at a site withinthe framework portion that undergoes a conformational change uponinteraction of the framework portion with an analyte.

In some instances, signaling by the signaling portion is detectablyaltered upon interaction (e.g., binding) of the framework portion withan analyte. For example, signaling by the signaling portion candetectably increase or detectably decrease upon interaction (e.g.,binding) of the framework portion with an analyte. In some cases,biosensors have a signal change upon binding (e.g., specific binding) totheir respective analyte of at least about, for example, ±0.5, and/or anincrease or decrease in signal of at least about, for example, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 250%, 500%, 750%, 1000%, ormore than 1000%, e.g., relative to unbound biosensor. In some increases,the level of signal change is linked to background signal. Valuesrepresented here can be converted and/or expressed into any conventionalunits using ordinary skill. For example, units can be expressed as‘signal change’ (as used above), ΔF/F and/or as signal-to-noise ratio(e.g., ΔF/F multiplied by the square root of the number of photonscollected). In some instances, signaling by a biosensor can be intensitybased.

In some instances, biosensors herein are distinguishable from Försterresonance energy transfer, also known as fluorescence resonance energytransfer (FRET)-based sensors, which require donor and acceptorchromophores, e.g., that function in concert, in that they includeindependently functioning or detectable signaling portions. For example,in some instances, signaling by a first signaling portion of a biosensorherein is independent of signaling by a second signaling portion withinthe same or a distinct biosensor. As noted above, signaling portions areallosterically regulated by the framework portion to which they arelinked upon interaction of the framework portion with an analyte (e.g.,a defined, selected, and/or specific analyte).

Framework Portions

Framework portions include genetically encoded macromolecules (e.g.,proteins or peptides) that undergo conformational alteration (e.g., astructural change) upon interaction (e.g., binding) with, or to, ananalyte (e.g., an analyte-binding dependent conformational alteration).For example, genetically encoded framework portions can have a firststructure in the absence of an analyte (e.g., in an unbound or openstate) and a second structure, that is detectably distinct (e.g.,differences in structures before and after a conformational change canbe observed using methods known in the art) from the first structure, inthe presence of an analyte (e.g., in a bound or closed state), e.g.,under physiologic conditions. In some instances, the conformationalchange that occurs upon interaction with an analyte (e.g., ananalyte-binding dependent conformational alteration) is detectablydistinct (e.g., can be observed using methods known in the art) from aconformational change that may occur for the same protein or peptideunder other physiological conditions (e.g., a change in conformationinduced by altered temperature, pH, voltage, ion concentration,phosphorylation).

Methods for identifying proteins or peptides that exhibit suitableconformational characteristics and/or for observing differences instructure between structures or before and after a conformational changeare known in the art and/or are described herein. Such methods caninclude, for example, one or more of structural analysis,crystallography, NMR, EPR using Spin label techniques, CircularDichroism (CD), Hydrogen Exchange surface Plasmon resonance,calorimetry, and/or FRET.

In some instances, framework portions can have a first structure in theabsence of an analyte (e.g., in an unbound or open state) and a secondstructure, that is detectably distinct (e.g., can be observed usingmethods known in the art) from the first structure, in the presence ofan analyte (e.g., in a bound or closed state), e.g., under physiologicconditions, wherein the structural change between the open and closedstate can allosterically modulate an independent signaling portionrecombinantly (e.g., artificially introduced) present within theframework portion (see, e.g., Structure I in FIG. 53).

Framework portions can also interact (e.g., bind) with at least oneanalyte (e.g., at least one defined, specific, and/or selected analyte).In some instances, a framework portion can interact specifically withone analyte (e.g., at least one defined, specific, and/or selectedanalyte). In such cases, affinity of binding between the frameworkbinding peptide and the analyte can be high or can be controlled (e.g.,with millimolar, micromolar, nanomolar, or picomolar affinity).Alternatively, the single framework binding protein can bind two or moreanalytes (e.g., two or more defined, specific, and/or selectedanalytes). In such cases, affinity of binding to the two or moreanalytes can be the same or distinct. For example, the affinity ofbinding can be greater for one analyte than it is for a second or third,etc., analyte. In some instances, binding between a framework portionand an analyte (e.g., at least one defined, specific, and/or selectedanalyte) have an affinity of for example, 10 mM to 1 pM.

As used herein, the term “analyte” can include naturally occurringand/or synthetic sugars, amino acids, proteins (e.g., proteins,peptides, and/or antibodies), hormones, ligands, chemicals (e.g., smallmolecules), pharmaceuticals, nucleic acids, cells, tissues, andcombinations thereof.

In some instances, biosensors can include one, two, or more frameworkbinding portions that bind (e.g., binds specifically) a single analyte(e.g., a single defined, specific, and/or selected analyte) or distinctanalytes (e.g., two or more distinct defined, specific and/or selectedanalytes). Alternatively or in addition, the framework portion can bechimeric. In such cases, a first part of the framework portion can be afirst peptide or can be derived from a first peptide, and a second partof the framework portion can be a second peptide or can be derived froma second peptide, wherein the first a second peptides are combined toresult in a single peptide.

Accordingly, framework portions can include macromolecules that undergoa conformational change upon interaction with an analyte. Onenon-limiting example of a suitable macromolecule is Calmodulin (CaM).CaM is in an extended shape in the absence of Ca²⁺ and in a condensedconformation in the presence of Ca²⁺ (Kuboniwa et al., Nat. Struc.Biol., 2:768-776, 1996 and Fallon and Quiocho, Structure, 11:1303-1307,2003).

In some instances, a framework binding portion can be a bacterialprotein or can be derived from a bacterial protein. Suitable bacterialproteins can include, but are not limited to, for example, periplasmicbinding proteins (PBPs).

PBPs from bacteria are generally useful in the biosensors herein atleast because they undergo dramatic conformational changes upon ligandbinding (Ouiocho et al. Mol. Microbiol., 20:17-225, 1996). X-ray crystalstructures of the apo (open) and bound (closed) forms of various PBPsreveal that these proteins have two (typically, although some have more)domains that undergo a large hinge-twist movement relative to each otherin a Venus flytrap manner (Dwyer and Hellinga, Curr. Opin. Struc. Biol.,14:495-504, 2004). This conformational change has been exploited tocreate a number of FRET-based genetically encoded sensors (see, e.g.,Deuschle et al., Pro. Sci, 14:2304-2314, 2005; Deuschle et al.,Cytometry, 64:3-9, 2005; Okumoto et al., Proc. Natl. Acad. Sci. USA.,102:8740-8745, 2005; Bogner and Ludewig, J. Fluoresc., 17:350-360, 2007;and Gu et al., FEBS Letters, 580:5885-5893, 2006). In addition, theligand-binding diversity of the PBP superfamily is large (Dwyer andHellinga, Curr. Opin. Struc. Biol., 14:495-504, 2004).

In some instances, framework portions can include, for example, one ormore of: arabinose-binding protein(s), glucose/galactose-bindingprotein(s), histidine-binding protein(s), maltose-binding protein(s),glutamine-binding protein(s), maltotriose-binding protein(s), RBP,ribose-binding protein(s), acetylcholine binding protein(s), cholinebinding protein(s), lysine binding protein(s), arginine bindingprotein(s), gamma aminobutyric acid (GABA) binding protein(s),ion-binding protein(s), peptide-binding protein(s), lactate-bindingprotein(s), histamine-binding protein(s), and/orLeucine/Isoleucine/Valine binding protein(s), including full lengthproteins, fragments, and/or variants thereof.

In some instances, exemplary framework portions can include: SEQ IDNO:105, which is Escherichia coli maltodextrin-binding protein (EcMBP)(UniProt accession number P0AEX9); SEQ ID NO: 106, which is PyrococcusFuriosus maltotriose-binding protein (PfMBP) (UniProt accession numberP58300); SEQ ID NO:107, which is E. coli glutamate-binding protein(EcYbeJ) (UniProt accession number Q1R3F7); SEQ ID NO:108, which is E.coli phosphonate-binding protein (EcPhnD) (UniProt accession numberP37902); and/or SEQ ID NO:109, which is Thermus thermophilusglucose-binding protein (TtGBP) (UniProt accession number Q72KX2,including full length proteins, fragments, and/or variants thereof.

In some instances, exemplary framework portions can include SEQ ID NO:110 (UniProt accession number Q92N37); SEQ ID NO:111 (UniProt accessionnumber D0VWx8, SEQ ID NO:112 (UniProt accession number Q7CX36), and/orSEQ ID NO:113 (UniProt accession number P0AD96, including full lengthproteins, fragments, and/or variants thereof.

In some instances, framework portions, or biosensors, do not includesignal peptides, or portions of signal peptides, that would otherwise bepresent in the peptide from which the framework portion is derived.

Signaling Portions

Biosensors herein include one or more genetically encoded signalingportions (e.g., independent signaling portions) within the amino acidsequence of a framework portion at a site(s) within the frameworkportion that undergo(es) a conformational change upon interaction of theframework portion with an analyte (e.g., a defined, specific, and/orselected analyte).

Signaling portions (e.g., independent signaling portions) includegenetically encoded molecules (e.g., peptides or proteins) that can beallosterically induced to emit a detectable signal (e.g., ananalyte-binding dependent signal).

In some instances, the detectable signal is detectably distinct (e.g.,can be distinguished using methods known in the art and/or disclosedherein) from a signal emitted by the molecule prior to allostericinducement (e.g., signaling portions can emit a detectable signal in twodetectably distinct states. For example, first signal can be emitted inunbound state and a second signal can be emitted in bound state). Asnoted above, in some instances, the detectable signal is proportional tothe degree of allosteric inducement. In some instances, if two or moresignaling portions are present in a biosensor, then two or moredetectably distinct signals can be emitted by the biosensor.

In some instances, a genetically encoded independent signaling portionis a genetically encoded fluorescent protein (FP), e.g., a macromoleculecontaining a functional group (e.g., a fluorophore) that absorbs energyof a specific wavelength and re-emits energy at a different (but equallyspecific) wavelength, including, for example, circularly permuted FP(cpFP).

As used herein, the term “fluorophore” relates to a functional group ina molecule which will absorb energy of a specific wavelength and re-emitenergy at a different (but equally specific) wavelength. In someinstances, fluorophore containing molecules include fluorescentproteins. The fluorophore in green fluorescent protein (GFP) includesSer-Tyr-Gly sequence (i.e., Ser65-dehydroTyr66-Gly67), which ispost-translationally modified to a4-(p-hydroxybenzylidene)-imidazolidin-5. Exemplary genetically encodedfluorescent proteins include, but are not limited to, fluorescentproteins from coelenterate marine organisms, e.g., Aequorea victoria,Trachyphyllia geoffroyi, coral of the Discosoma genus, Rennilla mulleri,Anemonia sulcata, Heteractis crispa, Entacmaea quadricolor, and/or GFP(including the variants S65T and EGFP, Rennilla mulleri GFP), cyanfluorescent protein (CFP), including Cerulean, and mCerulean3 (describedby Markwardt et al., PLoS ONE, 6(3)e17896.doi:10.1371/journal.pone.0017896), CGFP (CFP with Thr203Tyr: Hasan excitation and emission wavelength that is intermediate between CFPand EGFP), yellow fluorescent protein (YFP, e.g.,GFP-Ser65Gly/Ser72Ala/Thr203Tyr; YFP (e.g.,GFP-Ser65Gly/Ser72Ala/Thr203Tyr) with Val68Leu/Gln69Lys); Citrine (i.e.,YFP-Val68Leu/Gln69Met), Venus (i.e.,YFP-Phe46Leu/Phe64Leu/Met153Thr/Val163Ala/Ser175Gly), PA-GFP (i.e.,GFP-Val/163Ala/Thr203His), Kaede), red fluorescent protein (RFP, e.g.,long wavelength fluorescent protein, e.g., DsRed (DsRed1, DsRed2,DsRed-Express, mRFP1, drFP583, dsFP593, asFP595), eqFP611, and/or otherfluorescent proteins known in the art (see, e.g., Zhang et al., NatureReviews, Molecular and Cellular Biology, 3:906-908, 2002).

As set forth above, in some instances, fluorophore containing moleculesinclude fluorescent proteins that can be or that are circularlypermutated. Circular permutation methods are known in the art (see,e.g., Baird et al., Proc. Natl. Acad. Sci., 96:11241-11246, 1999; Topelland Glockshuber, Methods in Molecular Biology, 183:31-48, 2002).

In some instances, single-FP sensors have a number of advantages: theypreserve spectral bandwidth for multi-analyte imaging; their saturatedstates may be nearly as bright as the parental FP, and their ligand-freestates may be arbitrarily dim, providing large theoretical fluorescenceincreases. This allows for much greater changes in fluorescence and thusincreased signal-to-noise ratios and greater resistance tophotobleaching artifacts (Tian et al., Nat. Methods, 6:875-881, 2009).

In some instances, issues arising from long-term effects such as generegulation and protein expression and degradation can be identified bysimply fusing the intensity-based sensor to a another fluorescentprotein of different color, to serve as a reference channel.

In some instances, biosensors can include circularly permuted YFP(cpYFP) as a cpFP. cpYFP has been used as a reporter element in thecreation of sensors for H2O2 (HyPer) (Belousov et al., Nat. Methods,3:281-286, 2006), cGMP (FlincG) (Nausch et al., Proc. Natl. Acad. Sci.USA., 105:365-370, 2008), ATP:ADP ratio (Perceval) (Berg et al., Nat.Methods., 105:365-370, 2008), and calcium ions (Nakai et al., Nat.Biotechno., 19:137-141, 2001), including full length, fragments, and/orvariants thereof.

Linker Portions

As shown in Structure I (FIG. 53), biosensors herein can optionallyinclude one or more genetically encoded linkers positioned between oroperably linking the framework portion and the signaling portion. Linkerportions can include at least one naturally occurring or synthetic aminoacid (discussed below) as exemplified by SEQ ID NOs: 9-49, 54-61, 64-76,79-90, 95-104. In some instances, linker can include one or more of SEQID NOs: 9-49, 54-61, 64-76, 79-90, 95-104, and/or portions of SEQ IDNOs: 9-49, 54-61, 64-76, 79-90, 95-104. For example, linkers caninclude, but are not limited to, one or more of: PxSHNVY (SEQ IDNO:114), xPSHNVY (SEQ ID NO:115), xxSHNVY (SEQ ID NO:116), xxSHNVF (SEQID NO:117), PxSHNVF (SEQ ID NO:118), PxSYNVF (SEQ ID NO:119), xxSYNVF(SEQ ID NO:120), PxSYNVF (SEQ ID NO:121), xxSYNVF (SEQ ID NO:122),PxSxNVY (SEQ ID NO:123), PxSHxVY (SEQ ID NO:124), PxSHNxY (SEQ IDNO:125), PxSHNVx (SEQ ID NO:126), FNxxY (SEQ ID NO:127), FNxY (SEQ IDNO:128), FNY (SEQ ID NO:129), FxY (SEQ ID NO:130), xxY (SEQ ID NO:131),WxY (SEQ ID NO:132), xKY, (SEQ ID NO:133), FNPxY (SEQ ID NO:134), FNxPY(SEQ ID NO:135), HNS (SEQ ID NO:136), GGS (SEQ ID NO:137), xxS (SEQ IDNO:138), xxK (SEQ ID NO:139), GGK (SEQ ID NO:140), PXS (SEQ ID NO:141),xPS (SEQ ID NO:142), Px (SEQ ID NO:143), xP (SEQ ID NO:144), IxxS (SEQID NO:145), NxPK (SEQ ID NO:146), NPcK (SEQ ID NO:147), PPxSH (SEQ IDNO:148), PPxxSH (SEQ ID NO:149), PPPxSH (SEQ ID NO:150), PPxPSH (SEQ IDNO:151), xxSH (SEQ ID NO:152), PPxx (SEQ ID NO:153), FNxKN (SEQ IDNO:154), FNxxKN (SEQ ID NO:155), FNxPKN (SEQ ID NO:156), FNPxKN (SEQ IDNO:157), FNxx (SEQ ID NO:158), N, ADGSSH (SEQ ID NO:159), ADxxSH (SEQ IDNO:160), ADxPSH (SEQ ID NO:161), ADPxSH (SEQ ID NO:162), ADxx (SEQ IDNO:163), ADxxSH (SEQ ID NO:164), FNPG (SEQ ID NO:165), FNxxPG (SEQ IDNO:166), xxPG (SEQ ID NO:167), FNxx (SEQ ID NO:168), FNPx (SEQ IDNO:169), KYxxSH (SEQ ID NO:170), KYPxSH (SEQ ID NO:171), KYxPSH (SEQ IDNO:172), FxxP (SEQ ID NO:173), FNxP (SEQ ID NO:174), and/or FNPx (SEQ IDNO:175), where “x” indicates any amino acid.

Exemplary Biosensor Constructs

As noted above, biosensors herein include genetically encodedbiosensors, i.e., nucleic acids encoding biosensors, and/or the encodedbiosensors (e.g., isolated biosensors), for use as biosensors. In someinstances, nucleic acids encoding biosensors include isolated nucleicacids. In some instances, the portion of a nucleic acid encoding abiosensor can include a single reading frame encoding the biosensor. Forexample, a biosensor can be encoded by a portion of a nucleic acid thatfalls within a start codon and a stop codon. In some instances,biosensors are isolated (e.g., biosensors are substantially free ofcontaminating and/or non-biosensor components).

In some instances, biosensors can include, for example, one or moreframework portions selected from the group consisting of:arabinose-binding protein(s), glucose/galactose-binding protein(s),histidine-binding protein(s), maltose-binding protein(s),maltotriose-binding protein(s), glutamine-binding protein(s), RBP,ribose-binding protein(s), acetylcholine binding protein(s), cholinebinding protein(s), lysine binding protein(s), arginine bindingprotein(s), gamma aminobutyric acid (GABA) binding protein(s),ion-binding protein(s), peptide-binding protein(s), lactate-bindingprotein(s), histamine-binding protein(s), and/orLeucine/Isoleucine/Valine binding protein(s), including full lengthproteins, fragments, and/or variants thereof, including full lengthproteins, fragments and/or variants thereof, and at least oneindependent signaling portion present at a site within the frameworkportion that undergoes a conformational change upon interaction of theframework portion with an analyte.

In some instances, biosensors can include, for example, one or moreframework portions selected from the group consisting of: SEQ ID NO:105,which is Escherichia coli maltodextrin-binding protein (EcMBP) (UniProtaccession number P0AEX9); SEQ ID NO: 106, which is Pyrococcus Furiosusmaltose-binding protein (PfMBP) (UniProt accession number P58300); SEQID NO:107, which is E. coli glutamate-binding protein (EcYbeJ) (UniProtaccession number Q1R3F7); SEQ ID NO:108, which is E. coliphosphonate-binding protein (EcPhnD) (UniProt accession number P37902);and/or SEQ ID NO:109, which is Thermus thermophilus glucose-bindingprotein (TtGBP) (UniProt accession number Q72KX2), including full lengthproteins, fragments and/or variants thereof, and at least oneindependent signaling portion present at a site within the frameworkportion that undergoes a conformational change upon interaction of theframework portion with an analyte.

In some instances, biosensors can include, for example, one or moreframework portions selected from the group consisting of: SEQ ID NO: 110(UniProt accession number Q92N37); SEQ ID NO:111 (UniProt accessionnumber D0VWx8, SEQ ID NO:112 (UniProt accession number Q7CX36), and/orSEQ ID NO:113 (UniProt accession number P0AD96), including full lengthproteins, fragments and/or variants thereof, and at least oneindependent signaling portion present at a site within the frameworkportion that undergoes a conformational change upon interaction of theframework portion with an analyte.

In some instances, biosensors include any one or more:

Maltose biosensors SEQ ID NOs: 1-8 (i.e., Escherichia colimaltodextrin-binding protein (EcMBP)) or SEQ ID NOs: 50-53 (PyrococcusFuriosus maltose-binding protein (PfMBP)), including full lengthproteins, fragments and/or variants thereof;

Glutamate biosensors SEQ ID NOs: 62-63 (E. coli glutamate-bindingprotein (EcYbeJ)), including full length proteins, fragments and/orvariants thereof;

Phosphonate biosensors SEQ ID NOs: 77-78 (E. coli phosphonate-bindingprotein (EcPhnD)), including full length proteins, fragments and/orvariants thereof; and/or

Glucose biosensors SEQ ID NOs: 91-94 (Thermus thermophilusglucose-binding protein (TtGBP)), including full length proteins,fragments and/or variants thereof.

In some instances, nucleic acids encoding and/or amino acid sequences ofany of the framework portions, signaling portions, linker portions, orbiosensors (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53,62, 63, 77, 78, 91, 92, 93, and/or 94) (e.g., any amino acid sequence)disclosed herein can be modified to generate fragments (e.g., truncatedpeptides) and/or variants (e.g., peptides with a defined sequencehomology to the peptides disclosed herein). Variants can includeframework portions, signaling portions, linker portions, or biosensorswith amino acid sequences with homology to the framework portions,signaling portions, linker portions, or biosensors disclosed hereinand/or truncated forms of the framework portions, signaling portions,linker portions, or biosensors herein. In some instances, truncatedforms of the framework portions, signaling portions, linker portions, orbiosensors herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 50-100, 101-150, fewer amino acids than the framework portions,signaling portions, linker portions, and/or biosensors herein, e.g.,wherein the truncated biosensor variants retain at least at portion ofthe binding and/or signaling properties of same biosensor withouttruncation (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of thebinding and/or signaling properties of the same biosensor withouttruncation). In addition, truncations can be made at the amino-terminus,the carboxy-terminus, and/or within the body of the framework portions,signaling portions, linker portions, and/or biosensors herein.

While variants are generally observed and discussed at the amino acidlevel, the actual modifications are typically introduced or performed atthe nucleic acid level. For example, variants with 95%, 96%, 97%, 98, or99% sequence identity to SEQ ID NOs:91, 92, 93, and/or 94 can begenerated by modifying the nucleic acids encoding SEQ ID NOs: 91, 92,93, and/or 94 using techniques (e.g., cloning techniques) known in theart and/or that are disclosed herein.

As with all peptides, polypeptides, and proteins, including fragmentsthereof, it is understood that modifications to the amino acid sequencecan occur that do not alter the nature or function of the peptides,polypeptides, or proteins. Such modifications include conservative aminoacids substitutions and are discussed in greater detail below.

The peptides, polypeptides, and proteins, including fragments thereof,provided herein are biosensors whose activity can be tested or verified,for example, using the in vitro and/or in vivo assays described herein.

In some instances, any of the framework portions, signaling portions, orbiosensors (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53,62, 63, 77, 78, 91, 92, 93, and/or 94) (e.g., any amino acid sequence)described herein can be modified and varied so long as their desiredfunction is maintained. For example, the polypeptides can be modified aslong as the resulting variant polypeptides have the same or bettercharacteristics as the polypeptide from which they derived. For example,the variants can have the same or better affinity for their respectiveanalyte.

In some instances, the interacting face of a modified peptide can be thesame (e.g., substantially the same) as an unmodified peptide (methodsfor identifying the interacting face of a peptide are known in the art(Gong et al., BMC: Bioinformatics, 6:1471-2105 (2007); Andrade and Weiet al., Pure and Appl. Chem., 64(11):1777-1781 (1992); Choi et al.,Proteins: Structure, Function, and Bioinformatics, 77(1):14-25 (2009);Park et al., BMC: and Bioinformatics, 10:1471-2105 (2009)), e.g., tomaintain binding to an analyte. Alternatively, amino acids within theinteracting face can be modified, e.g., to decrease binding to ananalyte and/or to change analyte specificity.

The interacting face of a peptide is the region of the peptide thatinteracts or associates with other molecules (e.g., other proteins).Generally, amino acids within the interacting face are naturally morehighly conserved than those amino acids located outside the interactingface or interface regions of a protein. In some instances, an amino acidwithin the interacting face region of any of the framework portions orbiosensors (e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62,63, 77, 78, 91, 92, 93, and/or 94) (e.g., any amino acid sequence)disclosed herein can be the same as the amino acid shown in any of theframework portions or biosensors (e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94) (e.g., anyamino acid sequence) disclosed herein or can be include conservativeamino acid substitutions. In some instances, an amino acid within theinteracting face region any of the framework portions or biosensors(e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77,78, 91, 92, 93, and/or 94) (e.g., any amino acid sequence) disclosedherein can be substituted with an amino acid that increases theinteraction between the framework portion or biosensors (e.g, SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93,and/or 94) (e.g., any amino acid sequence) and an analyte.

In some instances, genetically encoded biosensors can include peptidesthat have at least 80, 85, 90, 95, 96, 97, 98, 99 percent identity tothe framework portions, signaling portions, or biosensors (e.g., SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93,and/or 94) (e.g., any amino acid sequence) described herein. Those ofskill in the art readily understand how to determine the identity of twopolypeptides. For example, the identity can be calculated after aligningthe two sequences so that the identity is at its highest level.

Another way of calculating identity can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Adv.Appl. Math, 2:482 (1981), by the identity alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of identity can be obtained for nucleic acids by, forexample, the algorithms disclosed in Zuker, Science 244:48-52 (1989);Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-10 (1989); Jaeger etal., Methods Enzymol. 183:281-306 (1989), which are herein incorporatedby reference for at least material related to nucleic acid alignment. Itis understood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity and to be disclosed herein.

Amino acid sequence modifications typically fall into one or more ofthree classes: substitutional, insertional, or deletional modifications.Insertions include amino and/or terminal fusions as well asintra-sequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of one to fourresidues. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. Amino acid substitutions are typically of singleresidues, but can occur at a number of different locations at once;insertions usually will be on the order of about from 1 to 10 amino acidresidues; and deletions will range about from 1 to 30 residues.Deletions or insertions can be made in adjacent pairs, i.e., a deletionof 2 residues or insertion of 2 residues. Substitutions, deletions,insertions or any combination thereof may be combined to arrive at afinal construct. The mutations must not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure. Substitutional modifications arethose in which at least one residue has been removed and a differentresidue inserted in its place. In some instances, substitutions can beconservative amino acid substitutions. In some instances, variantsherein can include one or more conservative amino acid substitutions.For example, variants can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, or 40-50 conservativeamino acid substitutions. Alternatively, variants can include 50 orfewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 9 or fewer, 8or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or2 or fewer conservative amino acid substitutions. Such substitutionsgenerally are made in accordance with the following Table 1 and arereferred to as conservative substitutions. Methods for predictingtolerance to protein modification are known in the art (see, e.g., Guoet al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004)).

TABLE 1 Conservative Amino Acid Substitutions Amino Acid Substitutions(others are known in the art) Ala Ser, Gly, Cys Arg Lys, Gln, His AsnGln, His, Glu, Asp Asp Glu, Asn, Gln Cys Ser, Met, Thr Gln Asn, Lys,Glu, Asp, Arg Glu Asp, Asn, Gln Gly Pro, Ala, Ser His Asn, Gln, Lys IleLeu, Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg, Gln, His Met Leu,Ile, Val, Ala, Phe Phe Met, Leu, Tyr, Trp, His Ser Thr, Cys, Ala ThrSer, Val, Ala Trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met, Ala, Thr

In some instances, substitutions are not conservative. For example, anamino acid can be replaced with an amino acid that can alter someproperty or aspect of the peptide. In some instances, non-conservativeamino acid substitutions can be made, e.g., to change the structure of apeptide, to change the binding properties of a peptide (e.g., toincrease or decrease the affinity of binding of the peptide to ananalyte and/or to alter increase or decrease the binding specificity ofthe peptide).

Modifications, including the specific amino acid substitutions, are madeby known methods. By way of example, modifications are made bysite-specific mutagenesis of nucleotides in the DNA encoding theprotein, thereby producing DNA encoding the modification, and thereafterexpressing the DNA in recombinant cell culture. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example M13 primer mutagenesis and PCRmutagenesis.

Nucleic Acids

The disclosure also features nucleic acids encoding the biosensors(e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77,78, 91, 92, 93, and/or 94) described herein, including variants and/orfragments of the biosensors (e.g., variants and/or fragments of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93,and/or 94). These sequences include all degenerate sequences related tothe specific polypeptide sequence, i.e., all nucleic acids having asequence that encodes one particular polypeptide sequence as well as allnucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the polypeptide sequences. Thus,while each particular nucleic acid sequence may not be written outherein, it is understood that each and every sequence is in factdisclosed and described herein through the disclosed polypeptidesequences.

In some instances, nucleic acids can encode biosensors with 95, 96, 97,98, or 99 identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52,53, 62, 63, 77, 78, 91, 92, 93, and/or 94.

In some instances, nucleic acids can encode SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 20-30, 30-40, or 40-50 conservative amino acidsubstitutions.

In some instances, nucleic acids can encode SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94containing 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 orfewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 orfewer, 3 or fewer, or 2 or fewer conservative amino acid substitutions

Also provided herein are vectors comprising the biosensors (e.g, SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93,and/or 94) described herein, including variants and/or fragments of thebiosensors (e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62,63, 77, 78, 91, 92, 93, and/or 94). For example:

Vectors can include nucleic acids that encode biosensors with 95, 96,97, 98, or 99 identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51,52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94.

Vectors can include nucleic acids that encode SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 20-30, 30-40, or 40-50 conservative amino acidsubstitutions.

Vectors can include nucleic acids that encode SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94containing 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 orfewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 orfewer, 3 or fewer, or 2 or fewer conservative amino acid substitutions

Examples of suitable vectors include, but are not limited to, plasmids,artificial chromosomes, such as BACs, YACs, or PACs, and viral vectors.As used herein, vectors are agents that transport the disclosed nucleicacids into a cell without degradation and, optionally, include apromoter yielding expression of the nucleic acid molecule in the cellsinto which it is delivered.

Viral vectors can include, for example, Adenovirus, Adeno-associatedvirus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNAviruses, including these viruses with the HIV backbone. Any viralfamilies which share the properties of these viruses which make themsuitable for use as vectors are suitable. Retroviral vectors, in generalare described by Coffin et al., Retroviruses, Cold Spring HarborLaboratory Press (1997), which is incorporated by reference herein forthe vectors and methods of making them. The construction ofreplication-defective adenoviruses has been described (Berkner et al.,J. Virology 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83(1986); Haj-Ahmad et al., J. Virology 57:267-74 (1986); Davidson et al.,J. Virology 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72(1993)). Recombinant adenoviruses have been shown to achieve highefficiency after direct, in vivo delivery to airway epithelium,hepatocytes, vascular endothelium, CNS parenchyma, and a number of othertissue sites. Other useful systems include, for example, replicating andhost-restricted non-replicating Vaccinia virus vectors.

Non-viral based vectors can include expression vectors comprisingnucleic acid molecules and nucleic acid sequences encoding polypeptides,wherein the nucleic acids are operably linked to an expression controlsequence. Suitable vector backbones include, for example, thoseroutinely used in the art such as plasmids, artificial chromosomes,BACs, YACs, or PACs. Numerous vectors and expression systems arecommercially available from such corporations as Novagen (Madison,Wis.), Clontech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), andInvitrogen/Life Technologies (Carlsbad, Calif.). Vectors typicallycontain one or more regulatory regions. Regulatory regions include,without limitation, promoter sequences, enhancer sequences, responseelements, protein recognition sites, inducible elements, protein bindingsequences, 5′ and 3′ untranslated regions (UTRs), transcriptional startsites, termination sequences, polyadenylation sequences, and introns.

Promoters controlling transcription from vectors in mammalian host cellsmay be obtained from various sources, for example, the genomes ofviruses such as polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis B virus, and most preferably cytomegalovirus(CMV), or from heterologous mammalian promoters, e.g. β-actin promoteror EF1α promoter, or from hybrid or chimeric promoters (e.g., CMVpromoter fused to the β-actin promoter). Of course, promoters from thehost cell or related species are also useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′ or3′ to the transcription unit. Furthermore, enhancers can be within anintron as well as within the coding sequence itself. They are usuallybetween 10 and 300 base pairs in length, and they function in cis.Enhancers usually function to increase transcription from nearbypromoters. Enhancers can also contain response elements that mediate theregulation of transcription. While many enhancer sequences are knownfrom mammalian genes (globin, elastase, albumin, fetoprotein, andinsulin), enhancers derived from a eukaryotic cell viruses can be used.Examples of such can include the SV40 enhancer on the late side of thereplication origin, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promoter and/or the enhancer can be inducible (e.g. chemically orphysically regulated). A chemically regulated promoter and/or enhancercan, for example, be regulated by the presence of alcohol, tetracycline,a steroid, or a metal. A physically regulated promoter and/or enhancercan, for example, be regulated by environmental factors, such astemperature and light. Optionally, the promoter and/or enhancer regioncan act as a constitutive promoter and/or enhancer to maximize theexpression of the region of the transcription unit to be transcribed. Incertain vectors, the promoter and/or enhancer region can be active in acell type specific manner. Optionally, in certain vectors, the promoterand/or enhancer region can be active in all eukaryotic cells,independent of cell type. Promoters of this type can include the CMVpromoter, the SV40 promoter, the β-actin promoter, the EF1α promoter,and the retroviral long terminal repeat (LTR).

The provided vectors also can include, for example, origins ofreplication and/or markers. A marker gene can confer a selectablephenotype, e.g., antibiotic resistance, on a cell. The marker product isused to determine if the vector has been delivered to the cell and oncedelivered is being expressed. Examples of selectable markers formammalian cells are dihydrofolate reductase (DHFR), thymidine kinase,neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin.When such selectable markers are successfully transferred into amammalian host cell, the transformed mammalian host cell can survive ifplaced under selective pressure. Examples of other markers include, forexample, the E. coli lacZ gene, green fluorescent protein (GFP), andluciferase. In addition, an expression vector can include a tag sequencedesigned to facilitate manipulation or detection (e.g., purification orlocalization) of the expressed polypeptide. Tag sequences, such as GFP,glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, orFLAG™ tag (Kodak; New Haven, Conn.) sequences typically are expressed asa fusion with the encoded polypeptide. Such tags can be insertedanywhere within the polypeptide including at either the carboxyl oramino terminus.

The disclosure further provides cells comprising the biosensors (e.g,SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91,92, 93, and/or 94) described herein, including variants and/or fragmentsof the biosensors (e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52,53, 62, 63, 77, 78, 91, 92, 93, and/or 94). Cells can include, forexample, eukaryotic and/or prokaryotic cells. For example, cells caninclude, but are not limited to cells of E. coli, Pseudomonas, Bacillus,Streptomyces; fungi cells such as yeasts (Saccharomyces, andmethylotrophic yeast such as Pichia, Candida, Hansenula, andTorulopsis); and animal cells, such as CHO, R1.1, B-W and LM cells,African Green Monkey kidney cells (for example, COS 1, COS 7, BSC1,BSC40, and BMT10), insect cells (for example, Sf9), human cells andplant cells. Suitable human cells can include, for example, HeLa cellsor human embryonic kidney (HEK) cells. In general, cells that can beused herein are commercially available from, for example, the AmericanType Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108. Seealso F. Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., (1998).

Optionally, the biosensors (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94) described herein,including variants and/or fragments of the biosensors (e.g., SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93,and/or 94) can be located in the genome of the cell (e.g., can be stablyexpressed in the cell) or can be transiently expressed in the cell.

Methods of making the provided cells are known and the method oftransformation and choice of expression vector will depend on the hostsystem selected. Transformation and transfection methods are described,e.g., in F. Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., (1998), and, as described above,expression vectors may be chosen from examples known in the art.

There are a number of compositions and methods which can be used todeliver the nucleic acid molecules and/or polypeptides to cells, eitherin vitro or in vivo via, for example, expression vectors. These methodsand compositions can largely be broken down into two classes: viralbased delivery systems and non-viral based deliver systems. Such methodsare well known in the art and readily adaptable for use with thecompositions and methods described herein.

By way of example, the provided polypeptides and/or nucleic acidmolecules can be delivered via virus like particles. Virus likeparticles (VLPs) consist of viral protein(s) derived from the structuralproteins of a virus. Methods for making and using virus like particlesare described in, for example, Garcea and Gissmann, Current Opinion inBiotechnology 15:513-7 (2004). The provided polypeptides can bedelivered by subviral dense bodies (DBs). DBs transport proteins intotarget cells by membrane fusion. Methods for making and using DBs aredescribed in, for example, Pepperl-Klindworth et al., Gene Therapy10:278-84 (2003). The provided polypeptides can be delivered by tegumentaggregates. Methods for making and using tegument aggregates aredescribed in International Publication No. WO 2006/110728.

Also provided are transgenic animals comprising one or more cells thebiosensors (e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62,63, 77, 78, 91, 92, 93, and/or 94) described herein, including variantsand/or fragments of the biosensors (e.g, SEQ ID NOs: 1, 2, 3, 4, 5, 6,7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94). As usedherein, the term animal refers to non-human animals, including, mammals,amphibians and birds. Specifically, examples include sheep, feline,bovines, ovines, pigs, horses, rabbits, guinea pigs, mice, hamsters,rats, non-human primates, and the like. As used herein, transgenicanimal refers to any animal, in which one or more of the cells of theanimal contain a heterologous nucleic acid. The heterologous nucleicacid can be introduced using known transgenic techniques. The nucleicacid is introduced into the cell, directly or indirectly. For example,the nucleic acid can be introduced into a precursor of the cell or byway of deliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The nucleic acid may be integratedwithin a chromosome, or it may be an extrachromosomally replicating DNA.

Methods for making transgenic animals using a variety of transgenes havebeen described in Wagner et al. (1981) Proc. Nat. Acad. Sci. USA,78:5016-5020; Stewart et al. (1982) Science, 217:1046-1048; Constantiniet al. (1981) Nature, 294:92-94; Lacy et al. (1983) Cell, 34:343-358;McKnight et al. (1983) Cell, 34:335-341; Brinstar et al. (1983) Nature,306:332-336; Palmiter et al. (1982) Nature, 300:611-615; Palmiter et al.(1982) Cell, 29:701-710; and Palmiter et al. (1983) Science,222:809-814. Such methods are also described in U.S. Pat. Nos.6,175,057; 6,180,849; and 6,133,502.

By way of example, the transgenic animal can be created by introducing anucleic acid into, for example, an embryonic stem cell, an unfertilizedegg, a fertilized egg, a spermatozoon or a germinal cell containing aprimordial germinal cell thereof, preferably in the embryogenic stage inthe development of a non-human mammal (more preferably in thesingle-cell or fertilized cell stage and generally before the 8-cellphase). The nucleic acid can be introduced by known means, including,for example, the calcium phosphate method, the electric pulse method,the lipofection method, the agglutination method, the microinjectionmethod, the particle gun method, the DEAE-dextran method and other suchmethod. Optionally, the nucleic acid is introduced into a somatic cell,a living organ, a tissue cell or other cell by gene transformationmethods. Cells including the nucleic acid may be fused with theabove-described germinal cell by a commonly known cell fusion method tocreate a transgenic animal.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g., mouse, rat,guinea pig, and the like. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of appropriate growthfactors, such as leukemia inhibiting factor (LIF). When ES cells havebeen transformed, they may be used to produce transgenic animals. Aftertransformation, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be detected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination or integration of the nucleic acid. Those colonies thatare positive may then be used for embryo manipulation and blastocystinjection. Blastocysts are obtained from 4 to 6 week old superovulatedfemales. The ES cells are trypsinized, and the modified cells areinjected into the blastocoel of the blastocyst. After injection, theblastocysts are returned to each uterine horn of pseudopregnant females.Females are then allowed to go to term and the resulting littersscreened for mutant cells having the construct. By providing for adifferent phenotype of the blastocyst and the ES cells, chimeric progenycan be readily detected. The chimeric animals are screened for thepresence of the nucleic acid, and males and females having themodification are mated to produce homozygous progeny transgenic animals.

Kits comprising one or more containers and the nucleic acid sequences,polypeptides, vectors, cells, provided herein, or combinations thereof,are also provided. For example, provided is a kit comprising (i) anucleic acid sequence encoding a biosensor described herein (e.g, one ormore of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77,78, 91, 92, 93, and/or 94), including variants and/or fragments of thebiosensor (e.g, variants or fragments of one or more of SEQ ID NOs: 1,2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or94), (ii) a polypeptide comprising a biosensor described herein (e.g,one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62,63, 77, 78, 91, 92, 93, and/or 94), including variants and/or fragmentsof the biosensor (e.g, variants or fragments of one or more of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93,and/or 94), (iii) a vector comprising the nucleic acid of (i), (iv) acell comprising the nucleic acid or (i) and/or the polypeptide of (ii),(v) a cell comprising the vector of (iii). The kit can comprise anycombination of (i)-(v). Optionally, the kit further comprises reagentsfor using the nucleic acid or peptide biosensors, vectors, and/or cells.For example, if the kit comprises cells, the kit may also comprise cellculture medium. Optionally, the kit further comprises instructions foruse. Optionally, the kit further comprises a GPCR, a GPCR-encodingnucleic acid sequence.

Design and Production/Manufacture Methods

Using the methods described herein, it is possible to design, produce,and/or adapt genetically encoded biosensors to assays for a variety ofclasses of analytes. The provided materials and methods facilitate thediscovery of new compounds targeting a wide array of protein targets,including but not limited to: endogenous targets responsible for diseasestate progression, targets on pathogens for treating infectiousdiseases, and endogenous targets to be avoided (thus screening early forpotential drug side effects and toxicity).

Methods herein provide systematic and generic approaches for the designand production of genetically encoded recombinant peptides containing ananalyte-binding framework portion linked (e.g., operably linked) to asignaling portion, wherein the signaling portion is allostericallymodulated or regulated by the framework portion upon interaction of theframework portion with an analyte. Generally, methods include: (i)selecting one or more target analytes; (ii) selecting a frameworkportion (e.g., a PBP) that interacts with (e.g., interacts specificallywith) or binds to (e.g., binds specifically to) the target analyte andthat undergoes a conformational change upon interacting with or bindingto the analyte; (iii) identifying sites or amino acid positions withinthe framework portion (e.g., the PBP) where the conformational changeoccurs; and (iv) inserting or cloning a signaling portion into the siteor amino acid position identified in (iii). Methods can, optionally,further include: (v) modifying or optimizing linker sequences betweenthe framework portion and the signaling portion, for example, by geneticmanipulation (e.g., by point mutation); (vi) modifying or optimizinganalyte binding; (vii) modifying the signal generated by the biosensor;and/or (viii) cloning the biosensor into a suitable vector.

In some instances: (iii) includes identification of insertion sites byanalysis of the structure (e.g., crystal structure) of the selectedframework portion (e.g., the selected PBP) in one or both of its openand closed states to determine amino acid positions at whichanalyte-binding dependent structural changes occur. In instances wherestructures for both open and closed states are not available, analysiscan be conducted by analogy to a structurally similar framework portion(e.g., PBP); (iv) includes cloning a signaling portion (e.g., a cpFP) atthe site identified in (iii) such that the analyte-binding dependentstructural change observed in (iii) will result in a conformationalchange in the signaling portion (e.g., the cpFP) and allostericmodulation of the signaling portion; (v) includes generating a libraryof mutants of biosensors with distinct linker sequences (e.g., by pointmutation), screening the library of mutants to identify mutants withenhanced properties (e.g., improved signal-to-noise ratio), andselecting mutants with enhanced properties (e.g., improvedsignal-to-noise ratio); (vi) includes increasing or decreasing bindingor affinity of the framework portion to the analyte, e.g., by modifyingamino acids in the interacting face of the framework portion or regionswithin the framework portion that are critical for analyte binding;(vii) includes increasing or decreasing signal emission by the signalingportion and/or changing the color of the signal where the signalingportion is a FP (e.g., a cpFP). Methods including (i)-(viii) areexemplified in the Examples section herein.

Methods of Use

The disclosure further provides methods for using the biosensorsdisclosed herein (e.g., one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 50, 51, 52, 53, 62, 63, 77, 78, 91, 92, 93, and/or 94), includingvariants and/or fragments of the biosensor (e.g., variants or fragmentsof one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, 53,62, 63, 77, 78, 91, 92, 93, and/or 94)) to detect analytes, e.g., inbiological systems. Such methods can include, for example:

Use of a maltose biosensor disclosed herein (e.g., one or more of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52, and/or 53 including variantsand/or fragments of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 50, 51, 52,and/or 53) to detect maltose, e.g., in a biological system;

Use of a glutamate biosensor disclosed herein (e.g., one or more of SEQID NOs: 62 and/or 63 including variants and/or fragments of SEQ ID NOs:62 and/or 63) to detect glutamate, e.g., in a biological system;

Use of a phosphonate biosensor disclosed herein (e.g., one or more ofSEQ ID NOs: 77 and/or 78 including variants and/or fragments of SEQ IDNOs: 77 and/or 78) to detect phosphonate, e.g., in a biological system;and/or

Use of a glucose biosensor disclosed herein (e.g., one or more of SEQ IDNOs: 91, 92, 93 and/or 94 including variants and/or fragments of SEQ IDNOs: 91, 92, 93 and/or 94) to detect glucose, e.g., in a biologicalsystem.

Techniques for performing such methods are known in the art and/or areexemplified herein. For example, methods can include introducing one ormore biosensors into a biological system (e.g., a cell); expressing theone or more biosensors in the biological system (e.g., the cell);monitoring the signal emitted by the expressed biosensor in thebiological system; and correlating the signal emitted by the expressedbiosensor in the biological system with a level of the analyte in thebiological system.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: Maltose Indicators

Genetically encoded maltose indicators were generated using Escherichiacoli maltodextrin-binding protein (EcMBP) as a framework and eithercircularly permuted β-lactamase (cpBla) or circularly permutedfluorescent protein (cpFP) as a signal. Data describe below suggest thatcpBla and cpFP are not interchangeable.

Allosteric coupling of ligand binding to fluorescence was hypothesizedto require:

i) that the site in into which cpGFP is inserted have the capacity totransduce the global conformational change the scaffold protein (EcMBPin this example) to the local environment of the chromophore in cpGFP;and

ii) that the local environment of the chromophore (e.g., linkers) beoptimized to maximize the difference in emission between unbound (apo)and the bound (in this example maltose-bound) states.

Example 1A: Identification of cpGFP Insertion Sites in EcMBP

Potential insertion sites were identified using the crystal structuresof the maltose-bound, closed form of EcMBP (Ouiocho et al., Structure,5:997-1015, 1997) and the ligand-free, open form of EcMBP shown in FIG.1 (Sharff et al., Biochemistry, 31:10657-10663, 1992) to guide rationaldesign of EcMBP-cpGFP fusions that would result in maltose-dependent GFPfluorescence.

For (i), the change in dihedral angle (defined by the Cα atoms spanningfour residues) was analyzed to identify maltose-dependent structuralchanges in sequentially adjacent residues (FIG. 6); this analysis showedthat the Cα chain is “torqued” around residues 175 (ΔDihedral=+41°) and311 (ΔDihedral=−22°) upon ligand binding. This sequential conformationalchange was predicted to be coupled to structural changes of an insertedcpGFP, resulting in maltose-dependent fluorescence for the fusionprotein.

Previous studies using randomly digested and reassembled circularlypermuted β-lactamase (cpBla) and EcMBP showed maltose-dependentβ-lactamase activity in proteins with insertions of cpBla at EcMBPresidues 165 and 317 (Guntas et al., Chem. Biol., 11:1483-1487, 2004;Guntas and Ostermeier, J. Mol. Biol., 336:263-273, 2004).

Since the ΔDihedral of EcMBP165 is +11° (moderate change) and EcMBP317is +2° (no real change), four EcMBP-cpGFP templates were constructed byinserting cpGFP into EcMBP at sites 165, 175 (identified herein), 311(identified herein), and 317 to test our predictive method and theinterchangeability of cpBla and cpGFP at sites identified from theEcMBP-cpBla screen. These constructs were named MBP165-cpGFP,MBP175-cpGFP, MBP311-cpGFP, and MBP317-cpGFP (names were modified toencompass variants (e.g., with modified linker sequences). The cpGFPused is cpGFP146 described in Baird et al. (Proc. Natl. Acad. Sci., USA,96:11241-11246, 1999). PCR assembly was used to construct fusionproteins with GlyGly-linkers between EcMBP and each terminus of cpGFP.The amino acid sequence of each construct is shown in FIGS. 6-9. Thesequences of SEQ ID NOs:1-3 shown in FIGS. 7A-7C (i.e., MBP165-cpGFP)differ in the linker sequence between MBP 1-165 and cpGFP 147-238(linker 1: see the line ending in amino acid 240)). The sequences of SEQID NOs: 4-5 shown in FIGS. 8A-8B (i.e., MBP175-cpGFP) differ in thesequence between MBP 1-175 and cpGFP 147-238 (linker 1: see the lineending in amino acid 240)). The sequences of SEQ ID NOs: 6-7 shown inFIGS. 9A-9B (i.e., MBP311-cpGFP) differ in the sequence between cpGFP1-146 and MBP 312-370 (linker 2: see the line ending in amino acid640)). Each construct includes 3 linkers: A linker between theC-terminus of the C-terminal portion of MBP and the N-terminus of cpGFP(i.e., linker 2), a linker between the N-terminus of cpGFP andC-terminus of the N-terminal portion of MBP, and a linker in cpGFP(i.e., linker 3).

Example 1B: Linker Optimization

Libraries of variants of SEQ ID NOs: 1-8 were generated with randomizedlinkers by single-stranded uracil template mutagenesis (see Kunkel etal., Methods Enzymol., 204:125-139, 1991) using the primers listedbelow:

165 Linker 1 Primers: (SEQ ID NO: 9) PLIAADGxxNVYIM (SEQ ID NO: 10)PLIAADxxNVYIM (SEQ ID NO11) PLIAADGGxxNVYIM (SEQ ID NO: 12)PLIAADGxPNVYIMG (SEQ ID NO: 13) PLIAADGIxNVYIMG (SEQ ID NO: 14)PLIAADPxSHNVYIM (SEQ ID NO: 15) PLIAADxPSHNVYIM (SEQ ID NO: 16)PLIAADxxSHNVYIM (SEQ ID NO: 17) PLIAADxxSHNVFIM (SEQ ID NO: 18)PLIAADPxSHNVFIM (SEQ ID NO: 19) PLIAADPxSYNVFIM (SEQ ID NO: 20)PLIAADxxSYNVFIM (SEQ ID NO: 21) PLIAADPxSYNVFIM (SEQ ID NO: 22)PLIAADxxSYNVFIM (SEQ ID NO: 23) PLIAADPxSxNVYIM (SEQ ID NO: 24)PLIAADPxSHxVYIM (SEQ ID NO: 25) PLIAADPxSHNxYIM (SEQ ID NO: 26)PLIAADPxSHNVxIM 165 Linker 2 Primers: (SEQ ID NO: 27) KLEYNFNxxYAFKYEN(SEQ ID NO: 28) KLEYNFNxYAFKYEN (SEQ ID NO: 29) KLEYNFNYAFKYEN(SEQ ID NO: 30) KLEYNFxYAFKYEN (SEQ ID NO: 31) KLEYNxxYAFKYEN(SEQ ID NO: 32) KLEYNWxYAFKYEN (SEQ ID NO: 33) KLEYNxKYAFKYEN(SEQ ID NO: 34) KLEYNFNPxYAFKYEN (SEQ ID NO: 35) KLEYNFNxPYAFKYEN175 Linker 1 Primers: (SEQ ID NO: 36) AFKYENxxSHNVYIM175 Linker 2 Primers: (SEQ ID NO: 37) KLEYNFNxxKYDIKDV311 Linker 1 Primers: (SEQ ID NO: 38) KSYEELxxSHNVYIM (SEQ ID NO: 39)KSYEELPxSHNVYIM (SEQ ID NO: 40) KSYEELxPSHNVYIM 311 Linker 2 Primers:(SEQ ID NO: 41) KLEYNFNxxAKDPRIA (SEQ ID NO: 42) KLEYNFNPxAKDPRIA(SEQ ID NO: 43) KLEYNFNxPAKDPRIA 317 Linker 1 Primers: (SEQ ID NO: 44)ELAKDPRxSHNVYIM (SEQ ID NO: 45) ELAKDPRxxSHNVYIM (SEQ ID NO: 46)ELAKDPRxxxSHNVYIM 317 Linker 2 Primers: (SEQ ID NO: 47) KLEYNFNxAATMENA(SEQ ID NO: 48) KLEYNFNxxAATMENA (SEQ ID NO: 49) KLEYNFNxxxAATMENA

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

About 400 variants were screened in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 10 mM maltose.

Insertion of cpGFP as MBP317, a site previously reported for cpBla, didnot show maltose-dependent fluorescence (FIG. 11) even though theframework protein still bound maltose, as determined by isothermaltitration calorimetry (FIG. 12). These data demonstrate thatidentification of insertion sites by a method other than insertion ofcpGFP (such as insertion of cpBla) is not sufficient to identify sitesthat transduce ligand binding to changes in fluorescence intensity

Insertion of cpGFP at residue 165 of EcMBP (EcMBP165-cpGFP), anotherposition reported in cpBla studies (Guntas and Ostermeier, supra) with-GlyGly-linkers flanking the cpGFP resulted in a protein in whichfluorescence increased 20% (ΔF/F=0.2) upon addition of saturatingmaltose.

Screening a fully-degenerate, length-two library (“XX”) at either theEcMBP-cpGFP linker (linker 1) or the cpGFP-EcMBP linker (linker 2)yielded proteins with maltose-dependent fluorescent increases >300% ordecreases >50% (FIG. 11). Many of the variants with increased ΔF/Fvalues had linkers containing proline(s). Subsequent librariesconstructed from oligonucleotides encoding XP or PX and randomization ofthe residues in GFP from residue 146 to 150 were screened, yielding afinal variant with: a two-proline EcMBP-cpGFP linker, a two-glycinecpGFP-EcMBP linker, GFP-H148Y, and GFP-Y151F. This variant, called“EcMBP165-cpGFP.PPYF” (abbreviated PPYF (SEQ ID NO:2)) has a ΔF/F=2.5, aKd for maltose of 3 μM. Screens also identified variantEcMBP311-cpGFP.L2-NP (-AsnPro- at linker 2 (SEQ ID NO:7)), which has aΔF/F of 1.0 and a Kd for maltose of 2 μM. This variant has an inferiormaltose-dependent fluorescence increase than PPYF, but demonstratesgenerality of the cpFP insertion method.

EcMBP175-cpGFP was also screened with XX linkers, and a few variantswith ΔF/F≈1 were identified (FIG. 11). One mutant, with the first linkerencoding HL (EcMBP175-cpGFP.L1-HL (SEQ ID NO:5)), has a ΔF/F=0.5 and aKd for maltose of 1.3 μM.

These data support that choice of insertion site by structural analysisis preferable to random insertion.

Example 1C: Modifying Ligand Binding and/or Fluorescent Properties ofSensors

One objective in the development of generic biosensors is for theframework to permit independent optimization of binding and signalingproperties. Analysis of whether biosensors herein permit suchoptimization was tested using the high-SNR sensor PPYF, by: (i)rationally altering maltose-binding affinity; (ii) changing theligand-binding specificity from maltose to sucrose, and (iii) creating afamily of sensors in multiple colors.

As a first step, the impacts of mutations of three tryptophanside-chains in the maltose-binding pocket (W230, W62, and W340) weretested. These sites have previously been shown to lower the affinity ofEcMBP for maltose by one, two, or three orders of magnitude,respectively, when mutated to alanine (Martineau et al., J. Mol. Biol.,214:337-352, 1990). A mutation to the hinge region, I329W, was also madeto PPYF, as this has been shown to increase maltose affinity by about2-fold in both wild-type EcMBP (Marvin and Hellinga, Nat. Struc. Biol.,8:795-798, 2001) and in the EcMBP-cpBla switches (Guntas et al., Chem.Biol., 11:1483-1487, 2004; Kim and Ostermeier, Arch. Biochem. Biophys.,446:44-51, 2006). As shown in FIG. 13, for the PPYF sensor, the threetryptophan-to-alanine binding-pocket mutations behaved as expected,lowering affinity by between one and three orders of magnitude. Incontrast, the I329W mutation did not increase affinity as expected, butrather decreased it. ΔF/F also decreased. This data suggests that themechanism of fluorescence change in this sensor is dependent on subtleinteractions between EcMBP and cpGFP that are linked to the I329Wmutation.

As an alternative test for changing the ligand-binding specificity ofthe sensor while preserving fluorescence signaling, “5-7” mutations(D14L, K15F, W62Y, E111Y), previously shown to confer EcMBP with anaffinity for sucrose (Guntas and Mansell, Proc. Natl. Acad. Sci.,102:11224-11229, 2005), were made to PPYF. As shown in FIG. 14A, themutations conferred to the sensor about 2 mM affinity for sucrose and ˜3mM affinity for maltose. To address a discrepancy between expected(micromolar) and observed (millimolar) affinity for disaccharides, the5-7 mutations were made to sensors with cpGFP inserted at differentpositions in EcMBP, and with different linker compositions. In thecontext of EcMBP165-cpGFP.PCF, the 5-7 mutations conferred very low (butobservable) binding preference for sucrose over maltose (FIG. 14B). Thetrend of higher (but still weak) affinity for sucrose (˜0.6 mM) overmaltose (˜6 mM) continued when the 5-7 mutations are made in the contextof EcMBP175-cpGFP.L1-HL (FIG. 14C). In the context ofEcMBP311-cpGFP.L2-NP, the 5-7 mutations appeared to eliminate allbinding (FIG. 14D). The preference for sucrose over maltose of the 5-7variants of the sensors is consistent with the binding properties of the5-7 variants of EcMBP alone and EcMBP-cpBla (Guntas and Mansell, Proc.Natl. Acad. Sci., 102:11224-11229, 2005). The lower affinity for bothligands of the 5-7 variants of the sensors may be the consequence of theinserted cpGFP shifting the open and closed equilibrium.

These data suggest that ligand binding and fluorescent properties ofbiosensors can be independently modified.

Example 1D: Modifying Sensor Color

The color of GFP can be altered by changing the amino acids that eithercomprise or interact with the chromophore (see Shaner et al., J. Cell.Sci. 120:4247-4260, 2007, for a review).

Using PPYF as a template, mutations Y66W (to yield a cyan variant,“cpCFP”), L64F+T65G+V68L+T203Y (yellow, “cpYFP”), and Y66H (blue,“cpBFP”) mutations were made (see Cubitt et al., Trends Biochem.,20:448-455, 1995, for exemplary methods). As shown in FIG. 15, thevariants exhibit fluorescence emission spectra consistent with theirrespective intended designs.

The ΔF/F of the color variants in response to maltose is different (ineach case inferior) from the ΔF/F of 2.5 observed in PPYF-green. TheEcMBP165-cpYFP.PPYF sensor, which has the same covalent chromophorestructure as PPYF, has the greatest ΔF/F of the three spectral variants(FIG. 15A). EcMBP165-cpCFP.PPYF has a lower ΔF/F than the green andyellow variants, but by incorporating previously identified mutations,(L1-PC+GFP-Y151F; the resulting protein is called EcMBP165.cpCFP.PCF), avariant with ΔF/F=0.8 was obtained (FIG. 15A).

The EcMBP165-cpBFP.PPYF variant, while dimly fluorescent, is not asensor, and a screen of 800 linker variants failed to produce anyvariant with ΔF/F>0.2 (FIG. 16).

Since EcMBP165-cpBFP.PPYF was very dim, Azurite mutationsT65S+V150I+V224R were included to increase brightness and stability, andmake EcMBP165-cpAzurite a good template for linker screening. Usingoligonucleotides encoding XX amino acid linkers, a variant was obtained,EcMBP165-cpAzurite.L2-FE, that had ΔF/F=0.8 (FIG. 15).

Example 1E: Modifying Sensor Color and Ligand Specificity/Affinity

The four sucrose-binding “5-7” mutations described above that conferredweak sucrose affinity in the green sensor (EcMBP165-cpGFP.PPYF) wereconverted to blue, cyan, and yellow maltose sensors(EcMBP165-cpAzurite.L2-FE, EcMBP165-cpCFP.PCF, and EcMBP165-cpYFP.PPYF).The green and yellow sensors showed increased fluorescence upon additionof 10 mM sucrose, but the cyan and blue proteins did not (FIG. 15A).Like the green variant, the yellow variant had no detectable sucroseaffinity with the wild type binding pocket (FIG. 15C) and millimolaraffinity for both sugars, with preference for sucrose over maltose (FIG.15D).

As seen in FIG. 17, as maltose concentration increased, the blue sensorincreased in fluorescence first (Kd ˜2.7 then the green (Kd ˜40 then theyellow (Kd ˜350 and at high maltose concentrations, the cyan variantbegan to increase its fluorescence (Kd ˜1.7 mM).

Example 1F: Imaging Bacteria

The ultimate value of genetically encoded fluorescent sensors is intheir utility for observing analyte flux in living cells and organisms.In a simple proof-of-principle experiment, Escherichia coli expressingPPYF or PPYF.T203V (see “Second-generation maltose sensors” below) wereimaged in the green fluorescence channel in the absence of maltose, andthen re-imaged after addition of saturating maltose to the media.

As shown in FIG. 18, bacteria expressing the sensors clearly becamebrighter, while control bacteria expressing EGFP appeared unchanged.Increased fluorescence was quantified by measuring the peak (gray-value)pixel intensity of each bacterium. Those expressing PPYF undergo anapproximate doubling of fluorescence (bacterium-averaged ΔF/F=1.1±0.4),those expressing PPYF.T203V have slightly increased ΔF/F(ΔF/F=1.29±0.2), while those expressing EGFP have no change influorescence (ΔF/F=−0.01±0.05).

Example 1F: 2-Photon Imaging of Mammalian Cells

Multi-photon microscopy opened new frontiers for in vivo fluorescenceimaging, in particular for neuronal activity visualization through theuse of genetically encoded calcium indicators (Tian et al., Nat.Methods, 3:281-286, 2009; Denk et al., Science, 248:73-76, 1990; Denkand Svoboda, Neuron, 18:351-357, 1997).

To demonstrate that the maltose sensors described herein have thepotential to be used for 2-photon imaging, fluorescence excitationspectra were collected. As shown in FIG. 19, with a 535 nm bandpassemission filter (50 nm bp), EcMBP165-cpGFP.PPYF showed a 10-foldmaltose-dependent increase in fluorescence when excited at 940 nm. Allfour spectral variants showed a significant maltose-dependent increasein 2-photon fluorescence.

Example 1G: Sub-Cloning Maltose Sensors

EcMBP165-cpGFP.PPYF.T203V (see “Second-generation maltose sensors”below) were cloned into a modified version of the pDisplay vector(Invitrogen) for extracellular display on the surface of transientlytransfected human embryonic kidney (HEK293) cells.

As shown in FIG. 20, the sensor localized to the plasma membrane andincreased in brightness in a concentration-dependent manner whenperfused with buffers of varying maltose concentration. The ΔF/F is5.8-fold, very close to that of the soluble protein produced in E. coli,with the mid-point of the maltose-dependent fluorescence increase being6.5 μM (FIG. 21A), very similar to the affinity determined on purifiedprotein (5 μM). Furthermore, the surface displayed sensor respondedrapidly to a pulse of 1 mM maltose (FIG. 21A), indicating that the timecourse for its action is useful for transient events.

Example 1H: Crystal Structure Analysis of Maltose Sensors

High-resolution structures of several of the maltose sensors describedabove were generated. Crystallization trials were performed withEcMBP165-cpGFP.PPYF, EcMBP175-cpGFP.L1-HL, and EcMBP311-cpGFP.L2-NP inthe presence and absence of excess maltose, from which bothEcMBP175-cpGFP.L1-HL and EcMBP311-cpGFP.L2-NP crystallized in thepresence of maltose. X-ray structures were solved to 1.9 and 2.0 Åresolution, respectively, by molecular replacement (FIGS. 22A-22C).

The structures of the cpGFP and EcMBP domains of the sensors aresuperimposable with published crystal structures of cpGFP (from GCaMP2;RMSD=0.36 and 0.38 Å, respectively, for comparing 221 common Cα atoms)and EcMBP-maltose (RMSD=0.43 and 0.37 Å, 370 Cα). The structure of EcMBPis largely unperturbed by insertion of the cpGFP domain; only residuesaround the 175 and 311 insertion sites showed any significantdisplacement.

GFP-H148, which H-bonds the GFP chromophore in the structure of nativeGFP, also directly H-bonded to the chromophore in theEcMBP175-cpGFP.L1-HL-maltose structure (FIG. 22B), although a differentrotamer was observed. In the EcMBP311-cpGFP.L2-NP-maltose structure,GFP-H148 is pulled away from the chromophore and is largely replaced bythe Asn from linker 2, which makes H-bond interactions to both strand 8of the GFP barrel and the chromophore phenolate oxygen (through a watermolecule, FIG. 22D). GFP-H148, meanwhile, seemed to stabilize theconformation of linker 2 of EcMBP311-cpGFP.L2-NP by H-bonding thebackbone carbonyl of the linker 2 Asn. There is some solvent access tothe cpGFP chromophore through the hole in the GFP barrel created bycircular permutation, although the inter-domain linkers block much ofthe opening in both structures. Relatively few contacts are made betweenthe cpGFP and EcMBP domains.

Based on the structures of two maltose-bound sensors, the sensingmechanism likely involves a shift in the relative position of linker 1and linker 2 induced by the conformational change in the EcMBP domainassociated with maltose binding (FIG. 5). The register shift ofinteractions between the two linkers could alter the proximity of linker2 and nearby side-chains to the cpGFP chromophore and change the waterstructure in the cpGFP opening, leading to a shift in the chromophoreprotonation equilibrium. This might explain why rigid proline ispreferred in either linker, since conformational changes upon ligandbinding might be better propagated through the rigid linkers to thecpGFP chromophore environment.

Example 1I: Generation of Second-Generation Maltose Sensors

In an attempt to increase brightness and ΔF/F of GCaMP, the localenvironment of the chromophore was altered by randomizing residueswithin cpGFP, and screening for improved variants (Tian et al., nat.Methods, 6:875-881, 2009).

As shown in FIG. 23, in the context of EcMBP165-cpGFP.PPYF, the T203Vmutation decreases the fluorescence emission of the apo-state by half(FIG. 23A), while saturated fluorescence and affinity are unchanged(FIG. 23B), increasing ΔF/F to 6.5. In the maltose-saturated state, PPYFitself has about a quarter the brightness of EGFP, and half thebrightness of cpGFP.

In the context of EcMBP311-cpGFP.L2-NP, the T203V mutation decreases thebrightness of both the apo-state and the saturated-state equally,resulting in no significant change in ΔF/F (FIGS. 23C and D).

These results indicate that the benefits of the T203V mutation are notuniversally transferable, and that cpGFP-based fluorescent sensors needto be optimized individually.

Example 2: Maltotriose Indicators

Genetically encoded maltotriose indicators were created using Pyrococcusfuriosus maltotriose binding protein. As described below, only thestructure of the ligand-bound state P. furiosus maltotriose bindingprotein (PfMBP) is available. As shown in FIGS. 1 and 2, PfMBP ishomologous to EcMBP (compare FIGS. 1 and 2). Two sensors were made,PfMBP171 and PfMBP316, the insertion points for which were selectedbased on homology to EcMBP165 and EcMBP311, respectively. Linkers wereoptimized. PfMBP sensors have a ΔF/F of ˜1.2.

Pyrococcus furiosus is a thermophilic organism. Proteins fromthermophiles have been shown to be more amenable to mutation than thosefrom mesophiles (Bloom et al., Proc. Natl. Acad. Sci., 103:5869-5874,2006). As an alternative to developing new sensors by inserting cpGFPinto PBPs, it should also be possible to generate new sensors bychanging the ligand-binding specificity of an existing PBP-based sensor.

It has previously been shown that the binding sites of PBPs can bereengineered to accommodate novel ligands (Looger et al., Nature,423:185-190, 2003). However, those re-design efforts used frameworkproteins from mesophiles and suffered from poor stability. Wehypothesized that PfMBP, which is intrinsically more stable than EcMBP,is more tolerant of mutations. To test this hypothesis, we characterizedand compared the stability of PfMBP to EcMBP, PfMBP-cpGFP sensors toEcMBP-cpGFP sensors, PfMBP binding site mutants to EcMBP binding sitemutants, and PfMBP-cpGFP sensor binding site mutants to EcMBP-cpGFPsensor binding site mutants. Conclusively, the PfMBP variants were morestable than the EcMBP variants. Finally, we demonstrate that theincreased thermo-stability of the PfMBP-cpGFP sensors is useful for themeasurement of maltotriose at temperatures as high at 60° C., whereasthe EcMBP-cpGFP sensors are only useful for the measurement of maltoseat temperatures as high as 40° C.

Example 2A: Identification of cpGFP Insertion Sites in PfMBP

The ligand-bound (closed) structure of PfMBP is available (Evdokimov etal., J. Mol. Biol., 305:891-904, 2001), but the unbound structure isnot. Accordingly, insertion sites for the PfMBP-cpGFP sensors wereidentified by homology to EcMBP.

Sites were selected based on the structural similarities between PfMBPand EcMBP. Two sites were selected. One of these sites is EcMBP311,which is homologous to PfMBP316. This site is at juncture between theend of the cluster of helices (Helices 8a, 8b, 8c) and the start of the“equatorial” spanning helix (Helix 9). Another site that was made into asensor in EcMBP was EcMBP165, which is homologous to PfMBP171. cpGFP wasinserted into PfMBP at each of these sites. The sequences of theresulting constructs, PfMBP171-cpGFP and PfMBP316-cpGFP, are shown inFIGS. 24 and 25, respectively.

Example 2B: Linker Optimization

Libraries of variants of SEQ ID NOs: 50-53 were generated withrandomized linkers by single-stranded uracil template mutagenesis usingthe primers listed below:

175 Linker 1 Primers: (SEQ ID NO: 54) AIAQAFxxSHNVYIMA (SEQ ID NO: 55)AIAQAFPxSHNVYIMA 171 Linker 2 Primers: (SEQ ID NO: 56) KLEYNFNxxYYFDDKTE316 Linker1 Primers (SEQ ID NO: 57) VLDDPExxHNVYIM (SEQ ID NO: 58)VLDDPEIxxSHNVYIM 316 Linker2 Primers (SEQ ID NO: 59) KLEYNFxxNDPVIY(SEQ ID NO: 60) KLEYNFNxPKNDPVIY (SEQ ID NO: 61) KLEYNFNPxKNDPVIY

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

Several thousand variants were screened in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 1 mM maltotriose.

Screening a fully-degenerate, length-two library (“XX”) at either thePfMBP171-cpGFP linker (linker 1) or the cpGFP-PfMBP linker (linker 2)yielded proteins with maltotriose-dependent fluorescent increases >100%or decreases >20% (FIG. 26A). A variant from this group with a GlyGlyPfMBP-cpGFP linker and a PheGlu cpGFP-PfMBP linker was selected forfurther characterization. This variant, called “PfMBP171-cpGFP.L2FE” hasa ΔF/F=1.2, a Kd for maltotriose of <1 μM.

Screening a fully-degenerate, length-two library (“XX”) at either thePfMBP316-cpGFP linker (linker 1) or the cpGFP-PfMBP linker (linker 2)also yielded proteins with maltotriose-dependent fluorescentincreases >100% or decreases >20% (FIG. 26B). A variant from this groupwith a GlyGly PfMBP-cpGFP linker and a PheGlu cpGFP-PfMBP linker wasselected for further characterization. This variant, called“PfMBP316-cpGFP.L1-NP” has a ΔF/F=1.2, a Kd for maltotriose of 40 μM.

These data support that structurally homologous frameworks can becompared to identify insertion sites for cpGFP.

Example 2C: Characterization of the Thermostability of the PfMBP andPfMBP-cpGFP Compared to EcMBP and EcMBP-cpGFP

Thermal stability of PfMBP171-cpGFP.L2FE was measured usingcircular-dichroism (CD) and compared to the original EcMBP and PfMBPbinding proteins, along with cpGFP. Following the changes by means of CDallowed determination of whether different transitions happened inalpha, beta, or both kinds of structures.

Given that cpGFP is a beta barrel, strong transitions in the beta signalalone were associated with changes in this kind of structure. In thesame way, transitions in both kinds of signals were associated with thebinding protein structure. As shown in FIG. 27A, PfMBP is significantlymore thermo-stable than EcMBP. In fact, while EcMBP denatured at about50° C., PfMBP did not denature at temperatures less than 80° C. Also,the addition of maltose to EcMBP stabilized the protein by about 10° C.

As shown in FIG. 27B, the stability of the EcMBP component of theEcMBP165-cpGFP.PPYF sensor decreased from 50° C. to 45° C. withinsertion of cpGFP, while the intrinsic stability of cpGFP in the sensorremained unchanged. There was little change in the stability of thePfMBP component of the PfMBP171-cpGFP.L2FE sensor with insertion ofcpGFP (FIG. 27B). Moreover, PfMBP seemed to exert a small stabilizingeffect over the inserted cpGFP, as shown by the change in the steepnessand melting point of the curve of the soluble form and thePfMBP171-cpGFP.L2FE sensor. All the associations made betweentransitions and domain unfolding were supported by CD spectra taken atthe beginning and the end of each temperature ramp.

Analysis of whether the PfMBP scaffold was more tolerant of mutationthan the EcMBP scaffold was also performed. Proof-of-principle mutationswere made to the ligand-binding sites of EcMBP and PfMBP, and theirrespective sensors. In EcMBP, Asn12 was mutated to Trp to result insteric clashes with the surrounding residues, and backbone, of thebinding pocket. The homologous mutation in PfMBP is Ala13Trp, whichwould be expected to have the same effect.

As shown in FIG. 27C, N12W decreased the Tm of EcMBP from 50° C. to 40°C., while the corresponding mutation in PfMBP, A13W, had no noticeableeffect. This data confirms that the thermo-philic protein is moretolerant of mutations to the binding site. Furthermore, in the contextof the sensors, the N12W mutation to EcMBP165-cpGFP.PPYF completelydestabilized the binding protein component of the sensor (FIG. 27D),while the A13W mutation in PfMBP171-cpGFP.L2FE had no effect onstability (FIG. 27D).

Example 2D: Tolerance of PfMBP Sensor to Increased Temperature

Fluorescence of the protein in the apo and ligand-bound states at wasmeasured at different temperatures.

As shown in FIG. 28A, fluorescence of the EcMBP165-cpGFP.PPYF sensor inthe bound state was higher than it is in the apo-state at lowertemperatures, by about 4-fold. However, at around 55° C. (the unfoldingtransition of the EcMBP component) the fluorescence of theEcMBP165-cpGFP.PPYF sensor dropped precipitously. As a result,EcMBP165-cpGFP.PPYF is unsuitable for detection of maltose attemperatures greater than 50° C. (FIG. 28B). In contrast,PfMBP171-cpGFP.L2FE retained its maltotriose binding capabilities athigh temperatures (FIGS. 28A and 28B), and is limited only by theintrinsic fluorescence of the cpGFP component, which decays at about 80°C. (FIG. 28A).

Example 2E: Measurement of Maltodextrins in Hot Liquids

To demonstrate that the soluble and immobilized sensors functionsimilarly, PfMBP171-cpGFP.L2FE, PfMBP316-cpGFPL1XXX, andEcMBP165-cpGFP.PPYF.T203V were immobilized via their N-terminalpoly-histidine tags on to the surface of Ni-NTA coated glass. In afluorescence plate reader, the immobilized proteins performed similarlyto their soluble counterparts (see FIGS. 28C, 28D, and 28F).

Next, a prototype device was constructed, with a light guide providingthe excitation light and returning the fluorescent emitted light back tothe photodetector, the bio-sensor protein immobilized to Ni-NTA coatedcoverslips, and the coverslip attached to the end of the light guide.The “wand” of the detector was dipped into different compositions ofsolutions, each with varying concentrations of maltose or maltotriose.Experiments were performed at different temperatures. PfMBP-cpGFP sensorperformed better at higher temperatures (as high as 60° C.) than theEcMBP-cpGFP sensor.

Example 3: Glutamate Indicators

Glutamate indicators were created from Escherichia coliglutamate-binding protein (EcYbeJ). As with PfMBP in Example 2, only thestructure of the ligand-bound EcYbeJ is available. EcYbeJ is homologousto EcMBP, but to a lesser degree. The best homology match between a sitein EcYbeJ and a site in a binding protein for which an intensity-basedsensor has already been created is EcYbeJ253 and EcMBP311 (describedherein). As shown in FIG. 3, both sites are at the junction of “RisingHelix 8” and the “Equatorial Helix/Coil.” The amino acid composition ofthe cpGFP and EcYbeJ junction was made the same as that of theEcMBP311-cpGFP sensor (Linker 2=NP). The amino acid composition of theEcYbeJ junction and cpGFP was optimized to LV (Linker 1=LV). The varianthas a ΔF/F of 5.

Example 3A: Identification of cpGFP Insertion Sites

The ligand-bound (closed) structure of Shigella flexneri glutamatebinding protein is available (Fan et al., Protein Pept. Lett.,13:513-516, 2006). This protein has only 4 amino acid mutations relativeto EcYbeJ, and is thus an appropriate model.

Insertion sites for the EcYbeJ-cpGFP sensors were identified by homologyto EcMBP. Based on the topology map (FIG. 3), position 311 in EcMBP wasidentified as an acceptable insertion site for EcYbeJ. EcMBP311 isequivalent to EcYbeJ253. EcYbeJ253 is at juncture between the end of thecluster of helices (Helices 8a, 8b, 8c) and the start of the“equatorial” spanning helix (Helix 9). In YbeJ, the structure that ishomologous to the equatorial helix is the equatorial coil (depicted inred, to match the red coloring of Helix 9).

Intrinsic affinity of wild-type YbeJ for glutamate (˜1 μM) was too highto permit high-throughput screening of linker libraries. Endogenousglutamate (from the growth media) saturates the sensor, makingmeasurement of the unbound state technically challenging. A mutation toYbeJ (A184V), in the “hinge” of the protein were made. Mutation of thisresidue to Trp or Arg have previously been shown to decrease affinity inFRET-based sensors (see Okumoto et al., Proc. Natl. Acad. Sci.,102:8740-8745, 2005). EcYbeJ253 (A184V)-cpGFP has an affinity forglutamate of about 100 μM. All references to EcYbeJ253-cpGFP, unlessotherwise noted, refer to the A184V variant. The sequences of the EcYbeJconstructs are shown in FIG. 29.

Example 3B: Linker Optimization

Libraries of variants of SEQ ID NOs: 62-63 were generated withrandomized linkers by single-stranded uracil template mutagenesis usingthe primers listed below:

253 Linker 1 Primers: (SEQ ID NO: 64) FKNPIPPxSHNVYIMA (SEQ ID NO: 65)FKNPIPPxxSHNVYIMA (SEQ ID NO: 66) FKNPIPPPxSHNVYIMA (SEQ ID NO: 67)FKNPIPPxPSHNVYIMA (SEQ ID NO: 68) KWFKNPIxxSHNVYIMA (SEQ ID NO: 69)FKNPIPPxxNVYIMAD (SEQ ID NO: 70) KWFKNPIxxNVYIMAD 253 Linker 2 Primers:(SEQ ID NO: 71) KLEYNFNxKNLNMNF (SEQ ID NO: 72) KLEYNFNxxKNLNMNF(SEQ ID NO: 73) KLEYNFNxPKNLNMNF (SEQ ID NO: 74) KLEYNFNPxKNLNMNF(SEQ ID NO: 75) GHKLEYNxxLNMNF (SEQ ID NO: 76) KLEYNFNxxLNMNF

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

Several thousand variants were screened in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 10 mM glutamate.

Screening a fully-degenerate, length-two library (“XX”) at theEcYbeJ253-cpGFP linker (linker 1) identified a sensor withglutamate-dependent fluorescent increases of ˜100%. This variant has aLeuVal EcYbeJ-cpGFP linker (L1-LV) and was used as the framework foroptimization of the cpGFP-EcYbeJ253 linker (linker 2). The results ofthat screen yielded a protein with glutamate-dependent fluorescentincrease of ˜500% and a linker 2 composition of AsnPro. As shown in FIG.30, this variant, called “EcYbeJ253-cpGFP.L1LVL2NP” has a ΔF/F=5, a Kdfor glutamate of 100 μM. Interestingly, the composition of the secondlinker, AsnPro, is the same as the linker composition ofEcMBP311-cpGFP.L2NP.

Example 3C: Detection of Extracellular Glutamate

EcYbeJ253-cpGFP.L1LVL2NP was cloned into the pDisplay™ vector to allowtargeting and anchoring of the sensor to the plasma membrane. Theresulting construct was transfected into cultured mammalian cells(HEK293) to visualize the addition of glutamate to extracellular media.Constructs were also generated in a bacterial expression vector with theepitope tags individually and in combination.

As shown in FIG. 31, the hemagglutinin tag interferes with thefluorescence change. EcYbeJ253-cpGFP.L1LVL2NP was re-cloned into aderivative of the pDisplay™ vector, lacking the hemagglutinin tag,called pMinDis (for Minimal Display). This new construct, when expressedin HEK293 cells, shows a change in fluorescence intensity under 2-photonexcitation that is approximately the same as the soluble protein (seeFIG. 32) with higher affinity, of about 1 (see FIG. 32).

To demonstrate that the sensor is functional in neurons, and not justcultured HEK cells, the gene from EcYbeJ253-cpGFP.L1LVL2NP was clonedinto an adeno-associated virus vector (AAV) under control of thesynapsin promoter. Virus particles were generated and used to infectcultured primary hippocampus neurons from rats 7 days after culturing.14 days after culturing (and 7 days after infection), the infectedneurons were imaged under 2-photon microscopy (FIG. 33).

Example 4: Phosphonate Indicators

An indicator for phosphonate compounds was created from Escherichia coliphosphonate-binding protein (EcPhnD). In this instance, only thestructure of the ligand-bound state was available at the time the sensorwas conceived. EcPhnD is homologous to EcMBP to a lesser degree and toEcYbeJ to a greater degree. The best homology match between a site inEcPhnD and a site in a binding protein for which an intensity-basedsensor has already been created is EcPhnD90 and EcYbeJ253. There is no“Rising Helix 8” in EcPhnD, but there is an “Equatorial Helix/Coil”(FIG. 4). cpGFP was inserted at the Equatorial Helix/Coil and linkerswere optimized to yield a sensor with ΔF/F of 1.2. EcPhnD is a dimmer,so, a pair of mutations (L297R+L301R) were made to convert it to amonomer. The monomer variant has a ΔF/F of 1.6.

Example 4A: Identification of cpGFP Insertion Sites in EcPhnD

Insertion sites for the EcPhnD-cpGFP sensors were identified using theligand-bound (closed) structure of EcPhnD by homology to EcMBP. Based onthe topology map (FIG. 4), position 311 in EcMBP was identified as anacceptable insertion site in EcPhnD. EcMBP311 corresponds to EcPhnD90.This site is at the point where the rising strand (Strand D) of EcPhnDhas a small bend in it that runs equatorial to the rest of the sheets inthe protein. Even though it is topologically different from the“equatorial” spanning helix (Helix 9) of EcMBP its equatorial alignmentis similar, and with just the closed structure at the time, in anenvironment that was expected to undergo significant dihedral changeupon binding ligand. Sequences of EcPhnD constructs are shown in FIG.34.

Example 4B: Linker Optimization

Libraries of variants of SEQ ID NOs: 77-78 were generated withrandomized linkers by single-stranded uracil template mutagenesis usingthe primers listed below:

90 Linker 1 Primers: (SEQ ID NO: 79) QTVAADGSSHNVYIMA (SEQ ID NO: 80)QTVAADxxSHNVYIMA (SEQ ID NO: 81) QTVAADxPSHNVYIMA (SEQ ID NO: 82)QTVAADPxSHNVYIMA (SEQ ID NO: 83) QTVAADxxNVYIMA (SEQ ID NO: 84)QTVAADxxSHNVYIMA (SEQ ID NO: 85) VFQTVAxxSHNVYIMA 90 Linker 2 Primers:(SEQ ID NO: 86) HKLEYNFNPGYWSVLI (SEQ ID NO: 87) HKLEYNFNxxPGYWSVLI(SEQ ID NO: 88) HKLEYNxxPGYWSVLI (SEQ ID NO: 89) HKLEYNFNxxYWSVLI(SEQ ID NO: 90) HKLEYNFNPxYWSVLI

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

Several thousand variants were screened in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 100 uM 2AEP.

Screening a number of fully-degenerate, libraries at the EcPhnD90-cpGFPlinker (linker 1) yielded a protein with 2AEP-dependent fluorescentincreases of >100%. This variant has a AlaAsp EcPhnD-cpGFP linker(L1-AD) and a ΔF/F of 1.2. The variant came from a linker that alsodeleted two residues, effectively making the insertion point of cpGFPoccur after residue D88, and then skipping to residue P91 at thecpGFP-EcPhnD linker.

It was observed from the crystal structure that EcPhnD forms a dimer. Todisrupt the dimer inter-face and potentially simplify the observablebinding behavior of the EcPhnD protein, two mutations, L297R and L301R,were introduced into the dimerization helices. These mutations wereexpected, by charge repulsion, to disrupt the dimer interface. As shownin FIG. 35, incorporation of L279R and L301R mutations intoEcPhnD90-cpGFP.L1AD caused ΔF/F to increases to 1.6 in response to 2AEP.

Further attempts to crystallize the open, ligand-unbound form of theprotein were successful after making a mutation to the binding site,H157A, that substantially decreased affinity for phosphonate compounds.This mutant was crystallized in the absence of ligand, and the openstate of the protein solved. The ΔDihedral analysis (FIG. 36) showedthat the region of greatest dihedral change was the group of residuesfrom 88-90, just one amino acid away from the site chosen by homology tothe equatorial helix.

These data further indicate that ΔDihedral metric is sufficient foridentifying sites in PBPs into which cpGFP can be inserted and result inintensity-based fluorescent sensors.

Example 5: Glucose Indicators

Glucose indicators were created from Thermus thermophilus glucosebinding protein (TtGBP). In this instance, only the structure of theligand-bound state is available. TtGBP is very homologous to EcMBP andPfMBP (compare FIG. 5 with FIGS. 1 and 2). The insertion point(TtGBP326) was chosen by homology to EcMBP311 and PfMBP316. The aminoacid composition of the cpGFP and TtGBP junction was made the same asthat of the EcMBP311-cpGFP and EcYbeJ253 sensors (Linker 2=NP). Linker 1was optimized (Linker 1=PA) and the TtGBP326 sensor have a ΔF/F of −2.5.To improve its utility for the measuring glucose concentrations in humanblood, the affinity was weakened from its native ˜1 μM to 1.5 mM bymutation of two residues in the binding pocket (H66A+H348A).

Example 5A: Identification of cpGFP Insertion Sites in TtGBP

The ligand-bound (closed) structure of TtGBP is available (Cuneo et al.,J. Mol. Biol., 362:259-270, 2006). Accordingly, insertion sites for theTtGBP-cpGFP sensors were identified by homology to EcMBP and PfMBP.Based on the topology map (FIG. 5), it is apparent that TtGBP, PfMBP,and EcMBP are structurally similar in the closed, ligand-bound state.Positions in EcMBP determined by the dihedral analysis (see above) werepredicted to be acceptable insertion sites in TtGBP. EcMBP311 ishomologous to TtGBP326. This site is at juncture between the end of thecluster of helices (Helices 8a, 8b, 8c) and the start of the“equatorial” spanning helix (Helix 9). The amino acid sequence of theTtGBP construct is shown in FIG. 37.

Example 5B: Linker Optimization

Libraries of variants of SEQ ID NO:91 were generated with randomizedlinkers by single-stranded uracil template mutagenesis using the primerslisted below:

326 Linker 1 Primers: (SEQ ID NO: 95) DSDPSKYxxSHNVYIM (SEQ ID NO: 96)DSDPSKYPxSHNVYIM (SEQ ID NO: 97) DSDPSKYxPSHNVYIM (SEQ ID NO: 98)RLDSDPSxxSHNVYIM (SEQ ID NO: 99) DSDPSKYxxNVYIM 326 Linker 2 Primers:(SEQ ID NO: 100) KLEYNFNxxNAYGQSA (SEQ ID NO: 101) KLEYNFxxPNAYGQSA(SEQ ID NO: 102) GHKLEYNxxNAYGQSA (SEQ ID NO: 103) KLEYNFNxPNAYGQSA(SEQ ID NO: 104) KLEYNFNPxNAYGQSA

Where “x” indicates that a degenerate primer (with DNA sequence “NNS”)was used to encode all 20 possible amino acids.

Several hundred variants were screen in semi-high-throughput fashion,measuring fluorescence intensity of clarified cell lysate in the absenceand presence of 10 mM glucose.

Linker 1 was optimized (Linker 1=PA) and the TtGBP326-cpGFP.L1PAL2NPsensor has a ΔF/F of ˜2.5 (see FIG. 38). Additionally, the TtGBP sensorwas tested with and without the N-terminal pRSET tag and no differencewas observed. Specifically, both sensors exhibited an affinity forglucose of about 1.5 mM and a ΔF/F of 2.5.

Data showing that it was possible to construct a glucose sensor byreplacing the EcMBP or PfMBP with TtGBP, retaining the composition oflinker 2, and optimizing the composition of linker 1, indicates that themethods for generating sensors disclosed herein can be used to generatesensors using any suitable framework.

Example 5C: Detecting Changes in Glucose Concentration In Vivo

The TtGBP326-cpGFP.L1PAL2NP sensor was cloned into a variant of thepDisplay™ vector lacking the N-terminal secretion sequence, theN-terminal hemagglutinin tag, the C-terminal cMyc tag, and theC-terminal PDGFR membrane anchoring domain.

The TtGBP sensor was cloned into a mammalian expression vector (based onthe pDisplay™ vector described in Example 3 above) with the secretion,epitope, and transmembrane anchoring peptides removed, thus resulting incytosolic expression of the TtGBP326-cpGFP.L1PAL2NP+H66A+H348A sensor.The construct was transfected into HEK293 cells. As shown in FIG. 39,the TtGBP sensor was expressed in the cytosol.

As shown in FIG. 40, addition of 10 mM glucose to the media increasesfluorescence.

The TtGBP326-cpGFP.L1PAL2NP+H66A+H348A sensor was further modified byL276V mutation to produce TtGBP326.L1PA.L2NP.H66A.H348A.L276V (see FIG.50). As shown in FIG. 51, this construct has an affinity for glucose of6.5 mM.

Additionally, the TtGBP326.L1P1.L2NP.G66A.H348A.L276V was cloned intothe pMinDis derivative of the pDisplay vector and expressed on theextracellular surface of HEK293 cells. After exchanging the HEK293 cellmedia for PBS, addition of glucose to the PBS led to an increase influorescence (see FIG. 52).

These data indicate, in part, that the pRSET tag is not essential to thefunction of the sensor and that the TtGBP326-cpGFP.L1PAL2NP sensor iscapable of detecting changes in the concentration of glucose inside oron the external surface of human cells.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A recombinant peptide biosensor comprising ananalyte-binding framework portion and a signaling portion, wherein thesignaling portion is present within the framework portion at a site oramino acid position that undergoes a conformational change uponinteraction of the framework portion with a defined, specific, orselected analyte, wherein the recombinant peptide biosensor comprises anamino acid sequence having at least 85% identity to a sequence selectedfrom the group consisting of SEQ ID NO: 77 and
 78. 2. The recombinantpeptide biosensor of claim 1, wherein the signaling portion isallosterically regulated by the framework portion such that signalingfrom the signaling portion is altered upon interaction of the frameworkportion with the analyte.
 3. The recombinant peptide biosensor of claim1, wherein signaling by the signaling portion detectably increases uponinteraction of the framework portion with the analyte.
 4. Therecombinant peptide biosensor of claim 1, wherein signaling by thesignaling portion detectably decreases upon interaction of the frameworkportion with the analyte.
 5. The recombinant peptide biosensor of claim1, wherein signaling by the signaling portion is proportional to thelevel of interaction between the framework portion and the analyte. 6.The recombinant peptide biosensor of claim 1, wherein the frameworkportion has a first structure in the absence of an analyte and a secondstructure, that is detectably distinct from the first structure, in thepresence of the analyte.
 7. The recombinant peptide biosensor of claim6, wherein the conformational change between the first structure and thesecond structure allosterically regulates the signaling portion.
 8. Therecombinant peptide biosensor of claim 1, wherein the framework portionis a periplasmic binding protein (PBP) or a variant of a PBP.
 9. Therecombinant peptide biosensor of claim 1, wherein the signaling portionis a circularly permuted fluorescent protein (cpFP).
 10. The recombinantpeptide biosensor of claim 1, wherein the analyte-binding frameworkportion binds specifically to phosphonate.
 11. A recombinant peptidebiosensor comprising an amino acid sequence with at least 95% identityto a recombinant peptide biosensor selected from the group consisting ofSEQ ID NO: 77 and 78, wherein the recombinant peptide biosensor bindsspecifically to phosphonate.
 12. The recombinant peptide biosensor ofclaim 11, comprising a recombinant peptide biosensor selected from thegroup consisting of SEQ ID NO: 77 and
 78. 13. A nucleic acid encodingthe recombinant peptide biosensor of claim
 1. 14. A nucleic acidencoding the recombinant peptide biosensor of claim
 11. 15. A vectorcomprising the nucleic acid of claim
 13. 16. A vector comprising thenucleic acid of claim
 14. 17. A cell comprising the nucleic acid ofclaim
 13. 18. A cell comprising the nucleic acid of claim
 14. 19. A cellcomprising the vector of claim
 15. 20. A cell comprising the vector ofclaim
 16. 21. A method for detecting phosphonate, the method comprisingdetecting a level of fluorescence emitted by the recombinant peptidebiosensor of claim 1, and correlating the level of fluorescence with thepresence of phosphonate.
 22. The method of claim 21, wherein therecombinant peptide biosensor is expressed from a nucleic acid.
 23. Themethod of claim 21, comprising contacting the recombinant peptidebiosensor with a sample comprising phosphonate.
 24. The method of claim21, comprising correlating the level of fluorescence with aconcentration of phosphonate.
 25. The method of claim 24, comprisingcomparing the level of fluorescence with a level of fluorescence emittedby the recombinant peptide biosensor in the presence of a samplecomprising a known concentration or range of concentrations ofphosphonate.
 26. A method for detecting phosphonate, the methodcomprising detecting a level of fluorescence emitted by a recombinantpeptide biosensor expressed from the nucleic acid of claim 13 andcorrelating the level of fluorescence with the presence of phosphonate.27. The method of claim 26, comprising contacting the recombinantpeptide biosensor with a sample comprising the phosphonate.
 28. Themethod of claim 27, comprising correlating the level of fluorescencewith a concentration of the phosphonate.
 29. The method of claim 28,comprising comparing the level of fluorescence with a level offluorescence emitted by the recombinant peptide biosensor in thepresence of a sample comprising a known concentration or range ofconcentrations of the phosphonate.